LOOF_2024_Annual_Report_Part3.pdf

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Attached to: Remedial Action Operations Support at Aberdeen Proving Ground 2026
Solicitation number: AU-2026-0000952
Issued by: Alabama

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Written by GovTribe AI · Feb 23, 2026

This is a quarterly biomonitoring report for the Old O-Field Groundwater Treatment Facility (GWTF) prepared as part of remedial action operations support at Aberdeen Proving Ground in Alabama, with Auburn University as the contracting entity. The 76th quarterly effluent toxicity study documents a 7-day cladoceran (Ceriodaphnia dubia) survival and reproduction test and a 7-day fathead minnow (Pimephales promelas) survival and growth test conducted on effluent samples collected during the week of September 9, 2024. Effluent samples were collected on September 9, 11, and 13, 2024, in 1-gallon plastic containers and shipped to Eurofins Arkansas in Little Rock for toxicity testing. The testing utilized samples collected on September 9 and 11, as the September 13 sample exceeded the 36-hour holding period requirement. Targeted chemical analyses including volatile organics, heavy metals, total suspended solids, turbidity, 1,4-dithiane, 1,4-oxathiane, and thiodiglycol were performed by CT Laboratories in Baraboo, Wisconsin on a 24-hour composite sample collected on September 11, 2024.

The biomonitoring evaluation followed EPA protocols for short-term chronic toxicity testing using EPA moderately hard synthetic water as dilution water. Test conditions for the cladoceran included static renewal testing at 25°C with daily solution renewal, 30-mL test chambers containing 15-mL test solutions, and organisms aged 5-6 days old. All test data, including water quality parameters, survival and reproduction results, growth measurements, and statistical analyses, were documented in attachments to the quarterly report, which serves as part of the ongoing remedial action monitoring requirements at the facility.

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76th Quarter Final Report

Old O-Field GWTF Quarterly Effluent Toxicity Study

1.0 INTRODUCTION

The 76th quarterly biomonitoring evaluation of the Old O-Field GWTF effluent included a 7-day cladoceran (Ceriodaphnia dubia) survival and reproduction test and a 7-day fathead minnow

(Pimephales promelas) survival and growth test. Targeted chemical analyses, included volatile organics were performed on an effluent grab sample; heavy metals, total suspended solids, turbidity, 1,4- dithiane, 1,4-oxathiane, and thiodiglycol were performed on a 24-hour composite sample of the effluent.

2.0 MATERIALS AND METHODS

2.1 Effluent Collection

The following effluent samples were collected the week of 9 September 2024:

• Sample was collected on 9 September 2024 (3 gallons)

• Sample was collected on 11 September 2024 (3 gallons). Sampling for the targeted chemical analyses was completed on the grab and 24-hour composite sample.

• Sample was collected on 13 September 2024 (4 gallons)

The effluent water for the toxicity testing was collected in 1 gallon plastic containers and was sent to Eurofins Arkansas (Little Rock, Arkansas). Due to a delayed shipment to the laboratory on 13

September 2024, the third sample was out of the 36-hour holding period and was not utilized.

Eurofins completed the 7-day testing with the excess effluent water collected on 9 and 11

September 2024.

Attachment 1 includes the following: 7-day cladoceran bioassay water quality data, survival and reproduction data (Test Method 1002.0); the 7-day fathead minnow bioassay water quality data, survival and growth data (Test Method 1000.0); and statistical analyses.

All targeted chemical analyses discussed in Section 1.0 were conducted on a split aliquot of the

24- hour composite sample completed on 11 September 2024, by CT Laboratories (Baraboo, Wisconsin) (Attachment 2).

2.2 Test Species

2.2.1 Cladoceran

The potential short-term chronic toxicity (7-day exposure) of the effluent to the cladoceran was determined by the static renewal method (solutions renewed daily) given in the Environmental

Protection Agency’s (EPA's) "Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms (4th ed.)" (USEPA, 2002). USEPA moderately hard synthetic water (MHSW) was used as the dilution water (USEPA, 2002). The test conditions for the cladoceran are summarized in Table 1.

The cladocerans were cultured at 25 °C with MHSW. The diet consisted of a mixture of yeast-cerophyl-trout chow (YCT) and the green alga, Selenastrum capricornutum, added to the cladoceran culture.

Table 1

Summary of Test Conditions and Test Acceptability Criteria for the Ceriodaphnia dubia Survival and

Reproduction Toxicity Test (Test Method 1002.0)

Test protocol: EPA-821-R-02-013 (USEPA, 2002)

Test type: Static renewal (every 24 h)

Temperature: 25 ºC ± 1 ºC

Light quality Ambient laboratory illumination

Light intensity: 50-100 foot candles at surface of water

Photoperiod: 16 hours light, 8 hours dark

Test chamber size: 30 mL

Test solution volume: 15 mL

Renewal of test solutions: Daily

Age of test organisms: <24 hours old at start of exposure

No. neonates per test chamber: 1 Assigned using blocking by known parentage

No. replicate chambers per concentration: 10

No. neonates per concentration: 10

Feeding regime: Fed YCT and Selenastrum capricornutum suspension daily at each 24-hour renewal

Cleaning: New disposable beakers

Aeration: None

Dilution water: MHSW

Test concentrations: 5 (100%, 56%, 32%, 18%, and 10%)

Dilution series: 0.5

Test duration: Until 60% or more of surviving control females have three broods in 7 days

(maximum 8 days) Endpoints: Survival and reproduction

Sampling and sample holding requirements: N/A

Sample volume required: N/A

Test acceptability criterion: 80% or greater survival of all control organisms and an average of 15 or more young per surviving female in the control solutions. 60% of surviving control females must produce three broods (required)

All neonates used in the 7-day survival and reproduction test were produced by cladocerans in culture that had released at least three broods. The initial age of the neonates in each treatment was <24 hours old. The tests were conducted in 30 milliliter (mL) disposable beakers containing

15 mL of test solution. All tests were conducted in an environmental chamber at 25 ± 1°C under a 16-hour light:8-hour dark photoperiod (fluorescent lights; 50-100 foot candles at the surface of the culture vessels). All test organisms were fed daily as described above at each 24-hour renewal.

Routine water quality was taken at the beginning and end of each 24-hour renewal.

2.2.2 Fathead Minnow

The potential short-term chronic toxicity (7-day exposure) of the effluent to the fathead minnow was determined by the static renewal method (solutions renewed daily) given in EPA's "Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to

Freshwater Organisms (4th ed.)" (USEPA, 2002). The test conditions for the fathead minnow are summarized in Table 2.

Fathead minnow larvae were obtained from in-house culture at Eurofins Arkansas. The fathead minnow larvae used in the 7-day survival and growth tests were <24 hour old at the start of the test. The tests were conducted in 500 mL disposable beakers containing 250 mL of test solution.

All test organisms were fed brine shrimp (Artemia sp.) nauplii <24 hours old twice per day. All tests were conducted at 25 ± 1 °C under a 16-hour light:8 hour dark photoperiod (fluorescent lights; 50-100 foot candles). Routine water chemistry was performed at the beginning and end of each renewal.

2.3 Test Endpoints, Test Acceptability Criteria, and Statistical Analyses

The endpoints for the 7-day chronic cladoceran test were survival and neonate production. The endpoints for the 7-day chronic fathead minnow test were survival and growth. The test acceptability criteria for the cladocerans were 80% or greater survival in the controls and an average of 15 of more young per surviving female in the controls (60% of surviving control females must produce three broods). The test acceptability criteria for the fathead minnow were

80% or greater survival in the controls and an average dry weight per surviving organism in the controls that equaled or exceeded 0.25 milligram (mg).

The data was analyzed using EPA method criteria and CETIS (v2.1.4) statistical software. The cladoceran neonate survival rate was analyzed using the Fisher Exact/Bonferroni-Holm Test and reproduction was tested using the Steel Many-One Rank Sum Test. The fathead minnow survival rate was analyzed using the Steel Many-One Rank Sum Test and biomass data was tested using the Dunnett Multiple Comparison Test (Attachment 1).

2.4 Quality Assurance/Quality Control

As part of Eurofins quality assurance/quality control program, both cladoceran neonates (<24 hours old) and fathead minnow larvae (<24 hours old) were exposed to the reference toxicant sodium chloride in MHSW to determine the 7-day Inhibition Concentration (IC25) that causes a 25% reduction in reproduction and biomass in the cladoceran and fathead minnow, respectively

(Attachment 1).

Table 2

Summary of Test Conditions and Test Acceptability Criteria for the Pimephales promelas Larval

Survival and Growth Toxicity Test (Test Method 1000.0)

Test protocol: EPA-821-R-02-013 (USEPA, 2002)

Test type: Static renewal (every 24 h)

Temperature: 25 ºC ± 1 ºC

Light quality: Ambient laboratory illumination

Light intensity: 50-100 foot candles

Photoperiod: 16 h light, 8 h dark

Test chamber size: 500 mL

Test solution volume: 250 mL

Renewal of test solutions: Daily (required)

Age of test organisms: Newly hatched larvae 24 hours old

No. larvae per test chamber: 10

No. replicate chambers per concentration: 4

No. larvae per test concentration: 40

Source of food: Newly hatched Artemia nauplii (<24 hours old)

Feeding regime: Fed twice daily (once in the morning, once in the evening

Cleaning: Siphon daily, immediately before test solution renewal

Aeration: None

Dilution water: MHSW

Test concentrations: 5 (100%, 56%, 32%, 18%, and 10%)

Dilution factor: 0.5

Test duration: 7 days

Endpoint: Survival and growth (dry weight)

Sampling requirements: N/A

Sample volume: N/A

Test acceptability criteria: 80% or greater survival in controls; average dry weight per surviving organism in control chamber equals or exceeds 0.25 mg

3.0 RESULTS AND DISCUSSION

The cladoceran controls met the test acceptability criteria of 80% or greater survival and an average of 15 or more young per surviving female in the controls. Survival was 100% in the controls and the average reproduction per control female was 33.5 neonates. Likewise, the fathead minnow met the test acceptability criteria of 80% or greater survival and an average dry weight per surviving organism in the controls of ≥0.25 mg dry weight. Larval survival was 100% in the controls. The mean biomass of the control fish was 0.714 mg dry weight.

The effluent did not affect the survival or reproduction of the cladocerans after the 7-day exposure.

Fathead minnow survival or growth were not affected after 7 days of exposure to the effluent

(Table 3).

Table 3

Summary of the Results of the Chronic Bioassays

Test Species 7-day Exposure

LC50 NOEC LOEC ChV IC25

Cladoceran >100% 100% >100% >100% >100%

Fathead minnow >100% 100% >100% >100% >100%

ChV = chronic value

IC25 = inhibition concentration 25 (concentration of toxicant that would cause a 25% reduction of test population)

LC50 = lethal concentration 50 (concentration of toxicant that would cause death in 50% of the test population)

LOEC = lowest observed effect concentration

NOEC = no observable effects concentration

4.0 REFERENCES

University of Maryland. 2023. Old O-Field Groundwater Treatment Facility Old O-Field Study

Area Operable Unit 1 Quarterly Effluent Toxicity Study (75th Quarter Task Report). Wye

Research and Education Center, Queenstown, MD.

USEPA. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and

Receiving Waters to Freshwater Organisms (4th ed.). E PA-821-R-02-013. Office of

Water, U.S. Environmental Protection Agency, Washington, DC.

Attachment 1

ANALYTICAL REPORT

PREPARED FOR

Attn: Helen Cavanagh

Environmental Chemical Corp.

1304 Governors Court

Suites 101 & 102 Abingdon, Maryland 21009

Generated 9/30/2024 8:38:32 AM

JOB DESCRIPTION

Chronic Biomonitoring

JOB NUMBER

192-14956-1

See page two for job notes and contact information.

Little Rock AR 72204 8600 Kanis Rd Eurofins Arkansas https://eol.et.eurofinsus.com/myEOL/

Eurofins Arkansas

Job Notes This report may not be reproduced except in full, and with written approval from the laboratory. The results relate only to the samples tested. For questions please contact the Project Manager at the e-mail address or telephone number listed on this page.

The test results in this report relate only to the samples as received by the laboratory and will meet all requirements of the methodology, with any exceptions noted. This report shall not be reproduced except in full, without the express written approval of the laboratory. All questions should be directed to the Eurofins Environment Testing South Central, LLC Project Manager.

Authorization

Generated

9/30/2024 8:38:32 AM

Authorized for release by Alexandria Wyllia, Project Management Assistant I Alexandria.Wyllia@et.eurofinsus.com Designee for John Overbey, Business Unit Manager john.overbey@et.eurofinsus.comjohn.overbey@et.eurofinsus.com (501)224-5060

Page 2 of 42 9/30/2024

Survival: NOEC LOEC ChV Growth: NOEC LOEC IC25 ChV 100 >100 >100 100 >100 >100 >100

Survival: NOEC LOEC ChV Reproduction: NOEC LOEC IC25 ChV 100 >100 >100 100 >100 >100 >100

Method 1002.0 Chronic Ceriodaphnia dubia Survival and Reproduction Test: The following were concluded from the test:

Re: Chronic Pimephales promelas (Fathead minnow) and Ceriodaphnia dubia Old O-Field - Maryland NPDES Permit No. NA Control No. 275227-1

This report is the analytical results and supporting information for the samples submitted to Eurofins Arkansas. The following results are applicable only to the sample identified by the control number referenced above. Accurate assessment of the data requires access to the entire document. Each section of the report has been reviewed and approved by the Laboratory Manager or qualified designee.

Testing procedures and Quality Assurance were in accordance with "Short Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms" EPA-821-R-02-013, Fourth Edition, October 2002.

The supporting chemistry data included with this report is intended for accessing the basic water quality of the effluent as required by this test method and is not intended to be utilized for discharge monitoring reports. Test results are summarized below:

Due to arriving outside of the 36hr holding time, the third sample was not utilized. Please review with your regulatory authority if this test is acceptable.

Method 1000.0 Chronic Pimephales promelas (Fathead minnow) Survival and Growth Test: The following were concluded from the test:

Page 3 of 42 9/30/2024

Table of Contents

I. Control Acceptance Criteria

II. Outlined Report

III. Data Analysis

IV. Standard Reference Toxicants

V. Organism History

VI. Results Summary Pimephales promelas (Fathead minnow) Ceriodaphnia dubia

Appendix (Data) Test 1000.0 Pimephales promelas (Fathead minnow) Survival and Growth Test 1002.0 Ceriodaphnia dubia Survival and Reproduction

Appendix (Reference Toxicant)

Appendix (Completed Data Sheets for DEQ) Pimephales promelas (Fathead minnow) Survival and Growth Pimephales promelas (Fathead minnow) Chemical Parameters Chart Ceriodaphnia dubia Survival and Reproduction Ceriodaphnia dubia Chemical Parameters Chart

Appendix (Summary)

Page 4 of 42 9/30/2024

I. Control Acceptance Criteria

Pimephales promelas (Fathead minnow) Method 1000.0

CRITERIA RESULTS PASS/FAIL

Control Survival > or = 80% 100 PASS

Control Growth > or = 0.25 mg per Surviving minnow 0.714 PASS

Control Growth CV < or = 40% * 2.56 PASS

Growth Minimum Significant Difference 12 to 30% 10.6 BELOW

Critical Dilution CV < or = 40% * 8.04 PASS

* EPA Region 6 Requirement

Ceriodaphnia dubia Method 1002.0

CRITERIA RESULTS PASS/FAIL

Control Survival > or = 80% 100 PASS

Control Reproduction > or = 15 per Surviving Female 33.5 PASS

Control CV < or = 40% per Surviving Female * 12.6 PASS

Reproduction Minimum Significant Difference 13 to 47% 13.8 PASS

Critical Dilution CV < or = 40% * 12.2 PASS

* EPA Region 6 Requirement

II. Outlined Report A. Introduction

1. Permit Number: NA

2. Test Requirements: Test Methods 1000.0 and 1002.0 B. Source of Effluent/Dilution Water:

1. Effluent Samples:

a. Sampling Point: Old O-Field

b. Chemical Data:

Analysis Sample 1 Sample 2

Dissolved oxygen (mg/l) 8.1 8.2 pH (standard units) 6.1 7.2

Alkalinity (mg/l as CaCO3) 39 40

Hardness (mg/l as CaCO3) 300 260

Conductivity (umhos/cm) 2100 2100

Residual Chlorine (mg/l) <0.05 <0.05

Ammonia as N (mg/l) <0.1 <0.1

2. Dilution Water Samples:

Analysis 192-14833-A-3 192-14783-A-1

Dissolved oxygen (mg/l) 8.3 8.1 pH (standard units) 6.2 7.5

Alkalinity (mg/l as CaCO3) 64 59

Hardness (mg/l as CaCO3) 80 86

Conductivity (umhos/cm) 310 310

Residual Chlorine (mg/l) <0.05 <0.05

Page 5 of 42 9/30/2024

C. Test Methods

1. Test methods used:

Short Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, EPA-821-R-02-013; test Methods 1000.0 and 1002.0, Fathead Minnow Survival and Growth and Ceriodaphnia dubia Survival and Reproduction.

2. Endpoint: No Observable Effects Concentration (NOEC)

3. Test Conditions:

Pimephales promelas (Fathead minnow) Survival and Growth Method 1000.0 Date & Time Test Initiated: September 10, 2024 at 1631 Date & Time Test Terminated: September 17, 2024 at 1445 Type & Volume of Test Chamber: 500 ml disposable beaker Volume of Sample: 250 ml Number of Organisms per replicate: 10 Number of Replicates per dilution: 4

Ceriodaphnia dubia Survival and Reproduction Method 1002.0 Date & Time Test Initiated: September 10, 2024 at 1556 Date & Time Test Terminated: September 17, 2024 at 1623 Type & Volume of Test Chamber: 30 ml disposable beaker Volume of Sample: 15 ml Number of Organisms per replicate: 1 Number of Replicates per dilution: 10

4. Source of test organisms: In-house culture

5. Test Temperature: 25 +/- 1 degree Celsius

D. Test Organisms

1. Scientific Name

a. Test 1000.0 Pimephales promelas

b. Test 1002.0 Ceriodaphnia dubia

III. Data Analysis The data was analyzed using EPA method criteria and CETIS statistical software.

Page 6 of 42 9/30/2024

IV. Standard Reference Toxicants Sodium chloride in synthetic moderately hard water.

Pimephales promelas (Fathead minnow) A chronic reference test was performed on August 13, 2024 at 1152 to August 20, 2024 at 1029 The results were as follows: (Control No. 275161-1.)

Survival LC-50: 3711 mg/l Growth IC-25: 1902 mg/l Growth PMSD: 7.47

Ceriodaphnia dubia A chronic reference test was performed on August 13, 2024 at 1128 to August 19, 2024 at 1318 The results were as follows: (Control No. 275161-2.)

Survival LC-50: 1881 mg/l Reproduction IC-25: 894 mg/l Reproduction PMSD: 14.5

V. Organism History

Pimephales promelas (Fathead minnow) Date: September 10, 2024 Age: <24 hours Source: In-house culture Water: Moderately hard synthetic Temperature: 25 deg.C

Ceriodaphnia dubia Date: September 10, 2024 Age: <24 hours Source: In-house culture Water: Moderately hard synthetic Temperature: 25 deg.C

Page 7 of 42 9/30/2024

Larvae are exposed in a static renewal system for seven days to different concentrations of effluent with dilution water. Test results are based on the survival and growth (weight) of the larvae.

VI. Results Summary Pimephales promelas, Fathead minnow Larval Survival and Growth Test -- Method 1000.0

Effluent concentrations for this test were 10 %, 18 %, 32 %, 56 %, 100 % in accordance with the NPDES permit.

The test was initiated on September 10, 2024 at 1631 and continued through September 17, 2024 at 1445.

Statistical analyses were performed on the observed data and the no observable effects concentrations (NOECs) were as follows:

a.) NOEC survival = 100 % effluent b.) NOEC growth = 100 % effluent

Summary of the 7-day Fathead Minnow Survival and Growth

Mean Growth (mg)Concentration Percent Survival 0.714Control 100

0.74410 % 97.5

0.74218 % 100

0.77032 % 100

0.76056 % 100

0.798100 % 100

10.00 18.00 32.00 56.00 100.00 Concentration (%)

S u rv iv a l

Page 8 of 42 9/30/2024

Neonates are exposed in a static renewal system to different concentrations of effluent with dilution water until 60% of surviving control organisms have three broods of offspring or a maximum of eight test days.

VI. Results Summary Ceriodaphnia dubia, Cladoceran Survival and Reproduction Test -- Method 1002.0

Effluent concentrations for this test were 10 %, 18 %, 32 %, 56 %, 100 % in accordance with the NPDES permit.

The test was initiated on September 10, 2024 at 1556 and continued through September 17, 2024 at 1623.

Statistical analyses were performed on the observed data and the no observable effects concentrations (NOECs) were as follows:

a.) NOEC survival = 100 % effluent b.) NOEC reproduction = 100 % effluent

Summary of the 7-day Ceriodaphnia dubia Survival and Reproduction Data

Concentration Mean ReproductionPercent Survival Control 33.5100 10 % 32.8100 18 % 28.690.0 32 % 32.4100 56 % 32.3100 100 % 30.1100

10.00 18.00 32.00 56.00 100.00 Concentration (%)

S u rv iv a l

Page 9 of 42 9/30/2024

Concentration Replicate

Appendix (Data): Test 1000.0

Pimephales promelas (Fathead Minnow) 7-Day Survival

Date and Time Test Initiated:

Date and Time Test Terminated:

Number of Survivors Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

September 10, 2024 at 1631 September 17, 2024 at 1445

Control A 10 10 10 10 10 10 10

B 10 10 10 10 10 10 10

C 10 10 10 10 10 10 10

D 10 10 10 10 10 10 10

10 % A 10 10 9 9 9 9 9

B 10 10 10 10 10 10 10

C 10 10 10 10 10 10 10

D 10 10 10 10 10 10 10

18 % A 10 10 10 10 10 10 10

B 10 10 10 10 10 10 10

C 10 10 10 10 10 10 10

D 10 10 10 10 10 10 10

32 % A 10 10 10 10 10 10 10

B 10 10 10 10 10 10 10

C 10 10 10 10 10 10 10

D 10 10 10 10 10 10 10

56 % A 10 10 10 10 10 10 10

B 10 10 10 10 10 10 10

C 10 10 10 10 10 10 10

D 10 10 10 10 10 10 10

100 % A 10 10 10 10 10 10 10

B 10 10 10 10 10 10 10

C 10 10 10 10 10 10 10

D 10 10 10 10 10 10 10

Page 10 of 42 9/30/2024

Concentration Replicate Weight of pan + fish

Total weight of fish (g)

Original # of fish

Mean dry weight (mg)

Appendix (Data): Test 1000.0

Pimephales promelas (Fathead Minnow) 7-Day Growth

Test Initiated:

Test Terminated:

Weight of pan

September 10, 2024 at 1631 September 17, 2024 at 1445

Control A .66479 .67207 0.00728 10 0.728

B .65466 .66171 0.00705 10 0.705

C .66197 .66890 0.00693 10 0.693

D .65947 .66678 0.00731 10 0.731

10 % A .65899 .66594 0.00695 10 0.695

B .66567 .67340 0.00773 10 0.773

C .66463 .67250 0.00787 10 0.787

D .66371 .67093 0.00722 10 0.722

18 % A .66945 .67727 0.00782 10 0.782

B .66465 .67232 0.00767 10 0.767

C .65471 .66208 0.00737 10 0.737

D .66048 .66731 0.00683 10 0.683

32 % A .66047 .66886 0.00839 10 0.839

B .65489 .66266 0.00777 10 0.777

C .65309 .66016 0.00707 10 0.707

D .66068 .66825 0.00757 10 0.757

56 % A .65707 .66485 0.00778 10 0.778

B .65747 .66485 0.00738 10 0.738

C .66053 .66789 0.00736 10 0.736

D .66062 .66852 0.00790 10 0.790

100 % A .65369 .66255 0.00886 10 0.886

B .65979 .66711 0.00732 10 0.732

C .65943 .66734 0.00791 10 0.791

D .65645 .66429 0.00784 10 0.784

Page 11 of 42 9/30/2024

Appendix (Data): Test 1002.0

Ceriodaphnia dubia Survival and Reproduction

Date and Time Test Initiated:

Date and Time Test Terminated:

September 10, 2024 at 1556 September 17, 2024 at 1623

Concentration: Control

Day No. of Adults

Young per Adult

No. of Young1 2 3 4 5 6 7 8 9 10

Replicate

1 0 0 0 0 0 0 0 0 0 0 0 10 0.00 2 0 0 0 0 0 0 0 0 0 0 0 10 0.00 3 0 0 0 0 0 0 0 0 0 0 0 10 0.00 4 5 6 7 4 6 4 7 6 5 6 56 10 5.60 5 13 12 11 14 12 13 9 13 10 11 118 10 11.8 6 0 0 0 0 0 8 11 0 12 17 48 10 4.80 7 18 19 19 17 17 4 0 19 0 0 113 10 11.3

TOTAL 36 37 37 35 35 29 27 38 27 34 335 10 33.5

Concentration: 10 %

Day No. of Adults

Young per Adult

No. of Young1 2 3 4 5 6 7 8 9 10

Replicate

1 0 0 0 0 0 0 0 0 0 0 0 10 0.00 2 0 0 0 0 0 0 0 0 0 0 0 10 0.00 3 0 0 0 0 0 0 0 0 0 0 0 10 0.00 4 5 3 5 6 5 6 5 4 6 5 50 10 5.00 5 8 11 10 12 9 12 13 9 12 11 107 10 10.7 6 0 0 0 0 0 0 0 0 17 0 17 10 1.70 7 17 18 20 17 17 16 15 17 0 17 154 10 15.4

TOTAL 30 32 35 35 31 34 33 30 35 33 328 10 32.8

Concentration: 18 %

Day No. of Adults

Young per Adult

No. of Young1 2 3 4 5 6 7 8 9 10

Replicate

1 0 0 0 0 0 0 0 0 0 0 0 10 0.00 2 0 0 0 0 0 0 0 0 0 0 0 10 0.00 3 0 0 0 0 0 0 0 0 0 0 0 10 0.00 4 4 4 4 5 4 5 6 3 4 5 44 10 4.40 5 10 8 9 12 11 10 0X 13 13 10 96 9 10.7 6 0 0 0 0 0 13 X 0 14 0 27 9 3.00 7 17 17 17 16 19 0 X 16 0 17 119 9 13.2

TOTAL 31 29 30 33 34 28 6 32 31 32 286 10 28.6

Page 12 of 42 9/30/2024

Appendix (Data): Test 1002.0

Ceriodaphnia dubia Survival and Reproduction

Date and Time Test Initiated:

Date and Time Test Terminated:

September 10, 2024 at 1556 September 17, 2024 at 1623

Concentration: 32 %

Day No. of Adults

Young per Adult

No. of Young1 2 3 4 5 6 7 8 9 10

Replicate

1 0 0 0 0 0 0 0 0 0 0 0 10 0.00 2 0 0 0 0 0 0 0 0 0 0 0 10 0.00 3 0 0 0 0 0 0 0 0 0 0 0 10 0.00 4 4 3 4 5 6 3 4 6 5 3 43 10 4.30 5 8 11 12 14 12 9 11 11 12 12 112 10 11.2 6 0 0 0 0 0 14 0 0 15 0 29 10 2.90 7 16 17 17 16 19 0 19 20 0 16 140 10 14.0

TOTAL 28 31 33 35 37 26 34 37 32 31 324 10 32.4

Concentration: 56 %

Day No. of Adults

Young per Adult

No. of Young1 2 3 4 5 6 7 8 9 10

Replicate

1 0 0 0 0 0 0 0 0 0 0 0 10 0.00 2 0 0 0 0 0 0 0 0 0 0 0 10 0.00 3 0 0 0 0 0 0 0 0 0 0 0 10 0.00 4 6 4 5 3 4 6 5 6 4 4 47 10 4.70 5 12 8 10 12 9 11 13 14 9 10 108 10 10.8 6 0 0 0 0 0 0 0 15 0 17 32 10 3.20 7 16 16 19 16 18 16 19 0 16 0 136 10 13.6

TOTAL 34 28 34 31 31 33 37 35 29 31 323 10 32.3

Concentration: 100 %

Day No. of Adults

Young per Adult

No. of Young1 2 3 4 5 6 7 8 9 10

Replicate

1 0 0 0 0 0 0 0 0 0 0 0 10 0.00 2 0 0 0 0 0 0 0 0 0 0 0 10 0.00 3 0 0 0 0 0 0 0 0 0 0 0 10 0.00 4 3 4 5 4 5 6 5 6 4 7 49 10 4.90 5 10 12 6 9 11 9 6 10 12 13 98 10 9.80 6 0 0 0 0 0 0 0 0 10 0 10 10 1.00 7 18 17 17 15 18 14 13 19 0 13 144 10 14.4

TOTAL 31 33 28 28 34 29 24 35 26 33 301 10 30.1

Page 13 of 42 9/30/2024

Appendix (Reference Toxicant): Test 1000.0

Chronic Reference Toxicant, Pimephales promelas (Fathead Minnow)

LC50 Survival Data

Log(LC50) Average Log(LC50) Lower Limit Upper Limit

3.25

3.30

3.35

3.40

3.45

3.50

3.55

3.60

3.65

1/ 3/

3/ 7/

4/ 4/

5/ 2/

6/ 1/

8/ 1/

9/ 5/

1/ 4/

3/ 5/

4/ 2/

6/ 4/

7/ 2/

L o g (L

IC25 Growth Data

Log(IC25) Average Log(IC25) Lower Limit Upper Limit

2.9

3.1

3.2

3.3

3.4

3.5

1/ 3/

3/ 7/

4/ 4/

5/ 2/

6/ 1/

8/ 1/

9/ 5/

1/ 4/

3/ 5/

4/ 2/

6/ 4/

7/ 2/

L o g (I

Page 14 of 42 9/30/2024

Appendix (Reference Toxicant): Test 1002.0

Chronic Reference Toxicant, Ceriodaphnia dubia

3.18

3.20

3.22

3.24

3.26

3.28

3.30

3.32

3/ 7/

4/ 4/

5/ 2/

6/ 1/

8/ 1/

1/ 4/

3/ 5/

4/ 2/

5/ 2/

6/ 4/

7/ 2/

LC50 Survival Data

Log(LC50) Average Log(LC50) Lower Limit Upper Limit

L o g (L

2.85

2.90

2.95

3.05

3.10

3.15

3.20

3/ 7/

4/ 4/

5/ 2/

6/ 1/

8/ 1/

1/ 4/

3/ 5/

4/ 2/

5/ 2/

6/ 4/

7/ 2/

IC25 Reproduction Data

Log(IC25) Average Log(IC25) Lower Limit Upper Limit

L o g (I

Page 15 of 42 9/30/2024

SUMMARY REPORTING FORMS

CHRONIC BIOMONITORING

Pimephales promelas (Fathead Minnow)

SURVIVAL AND GROWTH

Appendix (Summary): Test 1000.0

Dilution water used:

Date and Time Test Terminated: September 17, 2024 at 1445 Date and Time Test Initiated: September 10, 2024 at 1631

NPDES No.: NA

Permittee: ECC

DATA TABLE FOR SURVIVAL

Percent Survival in replicate chambers

Mean percent survivalEffluent

Conc. % A B C D 24 hr 48 hr 7 days CV% Control 100 100 100 100 100 100 100 0.00 10 % 90.0 100 100 100 100 100 97.5 5.13 18 % 100 100 100 100 100 100 100 0.00 32 % 100 100 100 100 100 100 100 0.00 56 % 100 100 100 100 100 100 100 0.00 100 % 100 100 100 100 100 100 100 0.00

DATA TABLE FOR GROWTH

Effluent Average dry weight, mg replicate chambers Mean dry Conc. % A B C D weight, mg CV% Control 0.728 0.705 0.693 0.731 0.714 2.56 10 % 0.695 0.773 0.787 0.722 0.744 5.79 18 % 0.782 0.767 0.737 0.683 0.742 5.89 32 % 0.839 0.777 0.707 0.757 0.770 7.09 56 % 0.778 0.738 0.736 0.790 0.760 3.63 100 % 0.886 0.732 0.791 0.784 0.798 8.04

CV = Coefficient of variation = standard deviation * 100 / mean

Page 16 of 42 9/30/2024

Appendix (Summary): Test 1000.0

CHRONIC TOXICITY SUMMARY FORM

Pimephales promelas (Fathead minnow)

CHEMICAL PARAMETERS CHART

PERMITTEE:

NPDES NO.:

CONTACT:

ANALYST:

Test Initiated: DATE:

Test Terminated: DATE:

TIME:

TIME:NA

GCX6, V6YL, B6YF, QGL9, WK7B

September 10, 2024 1631ECC

Helen Cavanagh

September 17, 2024 1445

DILUTION DAY DILUTION DAY

1 2 3 4 5 6 7 1 2 3 4 5 6 7 Control 10 %

D.O. Initial 8.3 8.3 8.1 8.3 8.0 8.1 8.5 D.O. Initial 8.5 8.2 8.0 8.2 8.1 8.3 8.6 Final 6.7 6.9 6.6 6.5 7.2 6.9 6.8 Final 7.0 6.9 6.4 6.1 7.1 6.6 6.6 pH Initial 6.2 6.9 7.5 7.9 6.3 7.0 6.6 pH Initial 6.2 6.9 7.5 8.0 6.4 7.0 6.6 Final 7.4 7.4 7.3 5.8 7.4 6.9 6.4 Final 7.4 7.4 7.3 5.7 7.4 6.9 6.4

DILUTION DAY DILUTION DAY

1 2 3 4 5 6 7 1 2 3 4 5 6 7 18 % 32 %

D.O. Initial 8.4 8.3 8.1 8.2 8.0 8.4 8.3 D.O. Initial 8.2 8.4 8.1 8.3 8.1 8.4 8.6 Final 7.1 6.9 6.2 6.1 7.0 6.9 6.6 Final 7.0 7.2 6.5 7.1 7.3 6.5 6.6 pH Initial 6.2 6.9 7.5 7.9 6.4 7.0 6.6 pH Initial 6.3 6.9 7.4 7.9 6.4 7.0 6.6 Final 7.4 7.3 7.3 5.8 7.4 7.0 6.4 Final 7.4 7.4 7.4 5.9 7.4 6.9 6.4

DILUTION DAY DILUTION DAY

1 2 3 4 5 6 7 1 2 3 4 5 6 7 56 % 100 %

D.O. Initial 8.0 8.4 8.0 7.9 7.8 8.4 8.5 D.O. Initial 8.1 8.8 8.2 8.5 7.6 9.0 8.8 Final 7.1 7.2 6.4 6.4 7.2 6.5 6.5 Final 7.2 7.0 6.5 6.1 7.2 6.6 6.6 pH Initial 6.2 6.8 7.4 7.9 6.4 6.9 6.5 pH Initial 6.1 6.6 7.2 7.8 6.3 6.2 6.4 Final 7.4 7.3 7.4 5.8 7.4 6.9 6.5 Final 7.4 7.2 7.3 5.7 7.3 6.9 6.4

Alkalinity Hardness Conductivity Chlorine Sample ID

39 300 2100 <0.05 OF-EFF-090924 09-SEP-24

40 260 2100 <0.05 OF-EFF-091124 11-SEP-24

Alkalinity Hardness Conductivity Chlorine Sample ID

64 80 310 <0.05 192-14833-A-3 59 86 310 <0.05 192-14783-A-1

Page 17 of 42 9/30/2024

SUMMARY REPORTING FORMS

CHRONIC BIOMONITORING

Ceriodaphnia dubia

SURVIVAL AND REPRODUCTION

Appendix (Summary): Test 1002.0

Dilution water used:

Date and Time Test Terminated: September 17, 2024 at 1623 Date and Time Test Initiated: September 10, 2024 at 1556

NPDES No.: NA

Permittee: ECC

PERCENT SURVIVAL

Percent Effluent Time of Reading Control 18 % 32 % 56 % 100 %10 % 24 hour 100 100 100 100 100100 48 hour 100 100 100 100 100100 7 day 100 90.0 100 100 100100

NUMBER OF YOUNG PRODUCED PER FEMALE @ 7 DAYS

Percent Effluent Replicates Control 18 % 32 % 56 % 100 %10 % A 36 31 28 34 3130 B 37 29 31 28 3332 C 37 30 33 34 2835 D 35 33 35 31 2835 E 35 34 37 31 3431 F 29 28 26 33 2934 G 27 6 34 37 2433 H 38 32 37 35 3530 I 27 31 32 29 2635 J 34 32 31 31 3333 Mean per Adult 33.5 28.6 32.4 32.3 30.132.8 Mean per Surviving Adult 33.5 31.1 32.4 32.3 30.132.8

CV % 12.6 6.11 11.1 8.64 12.26.06

CV = Coefficient of variation = standard deviation * 100 / mean (calculated based on young produced by surviving females)

Page 18 of 42 9/30/2024

Appendix (Summary): Test 1002.0

CHRONIC TOXICITY SUMMARY FORM

Ceriodaphnia dubia

CHEMICAL PARAMETERS CHART

PERMITTEE:

NPDES NO.:

CONTACT:

ANALYST:

Test Initiated: DATE:

Test Terminated: DATE:

GCX6, V6YL, B6YF, QGL9, WK7B

TIME:

1556ECC

Helen Cavanagh

September 17, 2024 1623

September 10, 2024

Control 10 %

DILUTION DAY DILUTION DAY

1 2 3 4 5 6 7 1 2 3 4 5 6 7 D.O. Initial D.O. Initial 8.5 8.2 8.0 8.2 8.1 8.3 8.68.3 8.3 8.1 8.3 8.0 8.1 8.5 Final Final 8.5 7.8 8.0 7.8 7.9 8.8 8.08.5 7.8 8.0 7.9 7.8 8.8 8.3 pH Initial pH Initial 6.2 6.9 7.5 8.0 6.4 7.0 6.66.2 6.9 7.5 7.9 6.3 7.0 6.6 Final Final 8.2 6.2 7.9 6.6 7.6 7.6 6.78.2 6.1 7.8 6.6 7.9 7.5 6.7

18 % 32 %

DILUTION DAY DILUTION DAY

1 2 3 4 5 6 7 1 2 3 4 5 6 7 D.O. Initial D.O. Initial 8.2 8.4 8.1 8.3 8.1 8.4 8.68.4 8.3 8.1 8.2 8.0 8.4 8.3 Final Final 8.3 7.8 8.0 7.8 8.2 8.7 8.28.4 7.8 8.0 7.8 8.3 8.8 8.3 pH Initial pH Initial 6.3 6.9 7.4 7.9 6.4 7.0 6.66.2 6.9 7.5 7.9 6.4 7.0 6.6 Final Final 8.2 6.3 8.0 6.6 7.9 7.6 6.88.2 6.3 8.0 6.6 8.0 7.6 6.8

56 % 100 %

DILUTION DAY DILUTION DAY

1 2 3 4 5 6 7 1 2 3 4 5 6 7 D.O. Initial D.O. Initial 8.1 8.8 8.2 8.5 7.6 9.0 8.88.0 8.4 8.0 7.9 7.8 8.4 8.5 Final Final 8.4 7.8 8.1 7.7 8.2 8.7 8.08.5 7.8 8.0 7.8 8.2 8.6 8.2 pH Initial pH Initial 6.1 6.6 7.2 7.8 6.3 6.2 6.46.2 6.8 7.4 7.9 6.4 6.9 6.5 Final Final 8.1 6.3 7.9 6.5 7.9 7.7 6.88.2 6.3 8.0 6.6 7.9 7.7 6.8

Alkalinity Hardness Conductivity Chlorine Sample ID

39 300 2100 <0.05 OF-EFF-090924 09-SEP-24

40 260 2100 <0.05 OF-EFF-091124 11-SEP-24

Alkalinity Hardness Conductivity Chlorine Sample ID

64 80 310 <0.05 192-14833-A-3 59 86 310 <0.05 192-14783-A-1

Page 19 of 42 9/30/2024

CETIS Summary Report Report Date: 24 Sep-24 10:25 (p 1 of 2)

Test Code/ID: 275227_CD / 15-9600-6131

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Batch ID: 06-6608-8075

Start Date: 10 Sep-24 15:56

Ending Date: 17 Sep-24 16:23

Test Type: Reproduction-Survival (7d)

Test Length: 7d 0h

Protocol: EPA/821/R-02-013 (2002) Diluent: Mod-Hard Synthetic Water

Brine:Species: Ceriodaphnia dubia

Source: In-House Culture

Analyst:

Age: <24Taxon: Branchiopoda

Sample ID: 14-1185-4139

Sample Date: 09 Sep-24 12:45

Receipt Date: 10 Sep-24 09:25

Code: 275227

Sample Age: 27h

Source: ECC

Station: OF-EFF

Client:

Project:

Material: POTW Effluent

CAS (PC):

Multiple Comparison Summary

NOEL LOEL TOELEndpointAnalysis ID Comparison Method PMSD✓ STU

100 >100 ---12-5523-8419 ---7d Survival Rate 1Fisher Exact/Bonferroni-Holm Test 1

100 >100 ---17-8677-6741 13.8%Reproduction 1Steel Many-One Rank Sum Test 1

95% LCL 95% UCL

Point Estimate Summary

EndpointAnalysis ID Point Estimate Method %Level✓ STU

LC50 >100 --- ---09-9377-5722 <1Linear Interpolation (ICPIN)7d Survival Rate 1

IC25 >100 --- ---11-0122-1766 <1Linear Interpolation (ICPIN)Reproduction 1

Test Acceptability

Attribute Test Stat DecisionAnalysis ID Endpoint Lower OverlapUpper

TAC Limits

109-9377-5722 0.8Control Resp7d Survival Rate Yes Passes Criteria<<

112-5523-8419 0.8Control Resp7d Survival Rate Yes Passes Criteria<<

33.511-0122-1766 15Control RespReproduction Yes Passes Criteria<<

33.517-8677-6741 15Control RespReproduction Yes Passes Criteria<<

0.137917-8677-6741 0.13PMSDReproduction Yes Passes Criteria0.47

Code Mean Min Max Std DevCount CV%Std ErrConc-%

7d Survival Rate Summary

95% LCL 95% UCL %Effect

0 1.0000 1.0000 1.0000 0.000010 0.00%0.00001.0000 1.0000 0.00%D

10 1.0000 1.0000 1.0000 0.000010 0.00%0.00001.0000 1.0000 0.00%

18 0.9000 0.0000 1.0000 0.316210 35.14%0.10000.6738 1.1260 10.00%

32 1.0000 1.0000 1.0000 0.000010 0.00%0.00001.0000 1.0000 0.00%

56 1.0000 1.0000 1.0000 0.000010 0.00%0.00001.0000 1.0000 0.00%

100 1.0000 1.0000 1.0000 0.000010 0.00%0.00001.0000 1.0000 0.00%

Code Mean Min Max Std DevCount CV%Std ErrConc-%

Reproduction Summary

95% LCL 95% UCL %Effect

0 33.5 27 38 4.22310 12.61%1.33530.48 36.52 0.00%D

10 32.8 30 35 1.98910 6.06%0.628931.38 34.22 2.09%

18 28.6 6 34 8.1410 28.46%2.57422.78 34.42 14.63%

32 32.4 26 37 3.59610 11.10%1.13729.83 34.97 3.28%

56 32.3 28 37 2.79110 8.64%0.882530.3 34.3 3.58%

100 30.1 24 35 3.66510 12.18%1.15927.48 32.72 10.15%

CETIS™ v2.1.4.8 x64 (005-847-680-2) QA:________Analyst:________Convergent Rounding (4 sf)

Page 20 of 42 9/30/2024

CETIS Summary Report Report Date: 24 Sep-24 10:25 (p 2 of 2)

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

CodeConc-%

7d Survival Rate Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

3E6E26028AC3F1FE591B06A9A0122509MD5:

0 D 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

10 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

18 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.0000 1.0000 1.0000 1.0000

32 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

56 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

100 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

CodeConc-%

Reproduction Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

4FD6BA5464E93276BB3F98AA4D644C71MD5:

0 D 36 37 37 35 35 29 27 38 27 34

10 30 32 35 35 31 34 33 30 35 33

18 31 29 30 33 34 28 6 32 31 32

32 28 31 33 35 37 26 34 37 32 31

56 34 28 34 31 31 33 37 35 29 31

100 31 33 28 28 34 29 24 35 26 33

CodeConc-%

7d Survival Rate Binomials

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

0 D 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

10 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

18 1/1 1/1 1/1 1/1 1/1 1/1 0/1 1/1 1/1 1/1

32 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

56 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

100 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

CETIS™ v2.1.4.8 x64 (005-847-680-2) QA:________Analyst:________Convergent Rounding (4 sf)

Page 21 of 42 9/30/2024

Report Date: 24 Sep-24 10:25 (p 1 of 2)CETIS Analytical Report

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Analyzed: 24 Sep-24 10:24 Endpoint: Reproduction CETIS Version: CETISv2.1.4Analysis ID: 17-8677-6741

Analysis: Nonparametric-Control vs Treatments Status Level: 1

Edit Date: 24 Sep-24 10:19 MD5 Hash: 4FD6BA5464E93276BB3F98AA4D644C71 Editor ID: 005-847-680-2

Sample ID: 14-1185-4139

Sample Date: 09 Sep-24 12:45

Receipt Date: 10 Sep-24 09:25

Code: 275227

Sample Age: 27h

Source: ECC

Station: OF-EFF

Client:

Project:

Material: POTW Effluent

CAS (PC):

Batch ID: 06-6608-8075

Start Date: 10 Sep-24 15:56

Ending Date: 17 Sep-24 16:23

Test Type: Reproduction-Survival (7d)

Test Length: 7d 0h

Protocol: EPA/821/R-02-013 (2002) Diluent: Mod-Hard Synthetic Water

Brine:Species: Ceriodaphnia dubia

Source: In-House Culture

Analyst:

Age: <24Taxon: Branchiopoda

Test Stat Critical P-Value Decision(α:5%)TiesvsControl Conc-%

Steel Many-One Rank Sum Test

P-Typedf

91.5 75 2 0.4046 Non-Significant Effect10Dilution Water 18 CDF

81 75 2 0.1222 Non-Significant Effect18 18 CDF

93.5 75 3 0.4745 Non-Significant Effect32 18 CDF

91.5 75 4 0.4046 Non-Significant Effect56 18 CDF

79 75 3 0.0904 Non-Significant Effect100 18 CDF

NOEL LOEL Tox UnitsTOELAlt HypData Transform PMSDMSDu

100 >100 13.79%1---C > TUntransformed 4.62

Attribute Test Stat Decision

Test Acceptability Criteria

Lower OverlapUpper

TAC Limits

15Control Resp Yes Passes Criteria33.5 <<

0.13PMSD Yes Passes Criteria0.1379 0.47

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision(α:5%)

Between 174.283 34.8567 5 1.711 0.1477 Non-Significant Effect

Error 1099.9 20.3685 54

1274.18 59Total

ANOVA Assumptions Tests

Attribute Test Test Stat Critical P-Value Decision(α:1%)

20.91 15.09 0.0008 Unequal VariancesBartlett Equality of Variance TestVariance

0.7996 0.9459 <1.0E-05 Non-Normal DistributionShapiro-Wilk W Normality TestDistribution

Mean Min MaxMedianCountCode Std ErrConc-% CV% %Effect95% LCL 95% UCL

Reproduction Summary

33.5 27 38350 10 1.335 12.61%30.48 36.52D 0.00%

32.8 30 353310 10 0.6289 6.06%31.38 34.22 2.09%

28.6 6 343118 10 2.574 28.46%22.78 34.42 14.63%

32.4 26 3732.532 10 1.137 11.10%29.83 34.97 3.28%

32.3 28 3731.556 10 0.8825 8.64%30.3 34.3 3.58%

30.1 24 3530100 10 1.159 12.18%27.48 32.72 10.15%

CodeConc-%

Reproduction Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

0 D 36 37 37 35 35 29 27 38 27 34

10 30 32 35 35 31 34 33 30 35 33

18 31 29 30 33 34 28 6 32 31 32

32 28 31 33 35 37 26 34 37 32 31

56 34 28 34 31 31 33 37 35 29 31

100 31 33 28 28 34 29 24 35 26 33

CETIS™ v2.1.4.8 x64 (005-847-680-2)Convergent Rounding (4 sf) QA:________Analyst:________

Page 22 of 42 9/30/2024

Report Date: 24 Sep-24 10:25 (p 2 of 2)CETIS Analytical Report

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Analyzed: 24 Sep-24 10:24 Endpoint: Reproduction CETIS Version: CETISv2.1.4Analysis ID: 17-8677-6741

Analysis: Nonparametric-Control vs Treatments Status Level: 1

Edit Date: 24 Sep-24 10:19 MD5 Hash: 4FD6BA5464E93276BB3F98AA4D644C71 Editor ID: 005-847-680-2

Graphics

Conc-%

0 D 10 18 32 56 100

R ep ro d u ct io n

Rankits

2.01.51.00.50.0-0.5-1.0-1.5-2.0 U n tr an s fo rm e d

-5

-10

-15

-20

CETIS™ v2.1.4.8 x64 (005-847-680-2)Convergent Rounding (4 sf) QA:________Analyst:________

Page 23 of 42 9/30/2024

CETIS Analytical Report Report Date: 24 Sep-24 10:25 (p 1 of 4)

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Analyzed: 24 Sep-24 10:24 Endpoint: 7d Survival Rate CETIS Version: CETISv2.1.4Analysis ID: 09-9377-5722

Analysis: Linear Interpolation (ICPIN) Status Level: 1

Edit Date: 24 Sep-24 10:19 MD5 Hash: 3E6E26028AC3F1FE591B06A9A0122509 Editor ID: 005-847-680-2

X Transform Y Transform Resamples Exp 95% CL

Linear Interpolation Options

MethodSeed

Linear Linear 1000 Yes Two-Point Interpolation1928598

Attribute Test Stat Decision

Test Acceptability Criteria

Lower OverlapUpper

TAC Limits

0.8Control Resp Yes Passes Criteria1 <<

Sample ID: 14-1185-4139

Sample Date: 09 Sep-24 12:45

Receipt Date: 10 Sep-24 09:25

Code: 275227

Sample Age: 27h

Source: ECC

Station: OF-EFF

Client:

Project:

Material: POTW Effluent

CAS (PC):

Batch ID: 06-6608-8075

Start Date: 10 Sep-24 15:56

Ending Date: 17 Sep-24 16:23

Test Type: Reproduction-Survival (7d)

Test Length: 7d 0h

Protocol: EPA/821/R-02-013 (2002) Diluent: Mod-Hard Synthetic Water

Brine:Species: Ceriodaphnia dubia

Source: In-House Culture

Analyst:

Age: <24Taxon: Branchiopoda

Point Estimates

Level 95% LCL 95% UCL% Tox Units 95% LCL 95% UCL

LC50 >100 --- --- <1 --- ---

Mean Min MaxCountCode ΣA/ΣB

Calculated Variate(A/B)

Conc-% CV% %Effect

7d Survival Rate Summary

Mean %Effect

Isotonic Variate

Median

1.0000 10/101.0000 1.00000 10D 0.00%0.00% 1.0000 0.00%1.0000

1.0000 10/101.0000 1.000010 10 0.00%0.00% 1.0000 0.00%1.0000

0.9000 9/100.0000 1.000018 10 10.00%35.14% 0.9750 2.50%1.0000

1.0000 10/101.0000 1.000032 10 0.00%0.00% 0.9750 2.50%1.0000

1.0000 10/101.0000 1.000056 10 0.00%0.00% 0.9750 2.50%1.0000

1.0000 10/101.0000 1.0000100 10 0.00%0.00% 0.9750 2.50%1.0000

CodeConc-%

7d Survival Rate Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

0 D 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

10 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

18 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.0000 1.0000 1.0000 1.0000

32 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

56 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

100 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

CodeConc-%

7d Survival Rate Binomials

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

0 D 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

10 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

18 1/1 1/1 1/1 1/1 1/1 1/1 0/1 1/1 1/1 1/1

32 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

56 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

100 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

CETIS™ v2.1.4.8 x64 (005-847-680-2) QA:________Analyst:________Convergent Rounding (4 sf)

Page 24 of 42 9/30/2024

CETIS Analytical Report Report Date: 24 Sep-24 10:25 (p 2 of 4)

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Analyzed: 24 Sep-24 10:24 Endpoint: 7d Survival Rate CETIS Version: CETISv2.1.4Analysis ID: 09-9377-5722

Analysis: Linear Interpolation (ICPIN) Status Level: 1

Edit Date: 24 Sep-24 10:19 MD5 Hash: 3E6E26028AC3F1FE591B06A9A0122509 Editor ID: 005-847-680-2

Graphics

Conc-%

1009080706050403020100

7d S u rv iv al

R a te

0.96

0.80

0.64

0.48

0.32

0.16

0.00

CETIS™ v2.1.4.8 x64 (005-847-680-2) QA:________Analyst:________Convergent Rounding (4 sf)

Page 25 of 42 9/30/2024

CETIS Analytical Report Report Date: 24 Sep-24 10:25 (p 3 of 4)

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Analyzed: 24 Sep-24 10:24 Endpoint: Reproduction CETIS Version: CETISv2.1.4Analysis ID: 11-0122-1766

Analysis: Linear Interpolation (ICPIN) Status Level: 1

Edit Date: 24 Sep-24 10:19 MD5 Hash: 4FD6BA5464E93276BB3F98AA4D644C71 Editor ID: 005-847-680-2

X Transform Y Transform Resamples Exp 95% CL

Linear Interpolation Options

MethodSeed

Linear Linear 1000 Yes Two-Point Interpolation2088207

Attribute Test Stat Decision

Test Acceptability Criteria

Lower OverlapUpper

TAC Limits

15Control Resp Yes Passes Criteria33.5 <<

Sample ID: 14-1185-4139

Sample Date: 09 Sep-24 12:45

Receipt Date: 10 Sep-24 09:25

Code: 275227

Sample Age: 27h

Source: ECC

Station: OF-EFF

Client:

Project:

Material: POTW Effluent

CAS (PC):

Batch ID: 06-6608-8075

Start Date: 10 Sep-24 15:56

Ending Date: 17 Sep-24 16:23

Test Type: Reproduction-Survival (7d)

Test Length: 7d 0h

Protocol: EPA/821/R-02-013 (2002) Diluent: Mod-Hard Synthetic Water

Brine:Species: Ceriodaphnia dubia

Source: In-House Culture

Analyst:

Age: <24Taxon: Branchiopoda

Point Estimates

Level 95% LCL 95% UCL% Tox Units 95% LCL 95% UCL

IC25 >100 --- --- <1 --- ---

Mean Min MaxCountCode

Calculated Variate

Conc-% CV% %Effect

Reproduction Summary

Mean %Effect

Isotonic Variate

Median

33.5 27 380 10D 0.00%12.61% 33.5 0.00%35

32.8 30 3510 10 2.09%6.06% 32.8 2.09%33

28.6 6 3418 10 14.63%28.46% 31.1 7.16%31

32.4 26 3732 10 3.28%11.10% 31.1 7.16%32.5

32.3 28 3756 10 3.58%8.64% 31.1 7.16%31.5

30.1 24 35100 10 10.15%12.18% 30.1 10.15%30

CodeConc-%

Reproduction Detail

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 Rep 7 Rep 8 Rep 9 Rep 10

0 D 36 37 37 35 35 29 27 38 27 34

10 30 32 35 35 31 34 33 30 35 33

18 31 29 30 33 34 28 6 32 31 32

32 28 31 33 35 37 26 34 37 32 31

56 34 28 34 31 31 33 37 35 29 31

100 31 33 28 28 34 29 24 35 26 33

CETIS™ v2.1.4.8 x64 (005-847-680-2) QA:________Analyst:________Convergent Rounding (4 sf)

Page 26 of 42 9/30/2024

CETIS Analytical Report Report Date: 24 Sep-24 10:25 (p 4 of 4)

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Analyzed: 24 Sep-24 10:24 Endpoint: Reproduction CETIS Version: CETISv2.1.4Analysis ID: 11-0122-1766

Analysis: Linear Interpolation (ICPIN) Status Level: 1

Edit Date: 24 Sep-24 10:19 MD5 Hash: 4FD6BA5464E93276BB3F98AA4D644C71 Editor ID: 005-847-680-2

Graphics

Conc-%

1009080706050403020100

R ep ro d u ct io n

32.0

28.0

24.0

20.0

16.0

12.0

8.0

4.0

0.0

CETIS™ v2.1.4.8 x64 (005-847-680-2) QA:________Analyst:________Convergent Rounding (4 sf)

Page 27 of 42 9/30/2024

CETIS Analytical Report Report Date: 24 Sep-24 10:25 (p 1 of 2)

Ceriodaphnia 7-d Survival and Reproduction Test Eurofins Arkansas

Analyzed: 24 Sep-24 10:24 Endpoint: 7d Survival Rate CETIS Version: CETISv2.1.4Analysis ID: 12-5523-8419

Analysis: STP 2xK Contingency Tables Status Level: 1

Edit Date: 24 Sep-24 10:19 MD5 Hash: 3E6E26028AC3F1FE591B06A9A0122509 Editor ID: 005-847-680-2

Sample ID: 14-1185-4139

Sample Date: 09 Sep-24 12:45

Receipt Date: 10 Sep-24 09:25

Code: 275227

Sample Age: 27h

Source: ECC

Station: OF-EFF

Client:

Project:

Material: POTW Effluent

CAS (PC):

Batch ID: 06-6608-8075

Start Date: 10 Sep-24 15:56

Ending Date: 17 Sep-24 16:23

Test Type: Reproduction-Survival (7d)

Test Length: 7d 0h

Protocol: EPA/821/R-02-013 (2002) Diluent: Mod-Hard Synthetic Water

Brine:Species: Ceriodaphnia dubia

Source: In-House Culture

Analyst:

Age: <24Taxon: Branchiopoda

NOEL LOEL Tox UnitsTOELAlt HypData Transform

100 >100 1---C > TUntransformed

Test Stat P-Value Decision(α:5%)vsControl Conc-%

Fisher Exact/Bonferroni-Holm Test

P-Type

1.0000 1.0000 Non-Significant Effect10Dilution Water Exact

0.5000 1.0000 Non-Significant Effect18 Exact

1.0000 1.0000 Non-Significant Effect32 Exact

1.0000 1.0000 Non-Significant Effect56 Exact

1.0000 1.0000 Non-Significant Effect100 Exact

Attribute Test Stat Decision

Test Acceptability Criteria

Lower OverlapUpper

TAC Limits

0.8Control Resp Yes Passes Criteria1 <<

RNR NR + RCodeConc-% Prop NR Prop R %Effect

7d Survival Rate Frequencies

010 100 1.0000 0.0000 0.00%D

010 1010 1.0000 0.0000 0.00%

19 1018 0.9000 0.1000 10.00%

010 1032 1.0000 0.0000 0.00%

010 1056 1.0000 0.0000 0.00%

010 10100 1.0000 0.0000 0.00%

Mean Min MaxMedianCountCode Std ErrConc-% CV% %Effect95% LCL 95% UCL

7d Survival Rate Summary

1.0000 1.0000 1.00001.00000 10 0.0000 0.00%1.0000 1.0000D 0.00%

1.0000 1.0000 1.00001.000010 10 0.0000 0.00%1.0000 1.0000 0.00%

0.9000 0.0000 1.00001.000018 10 0.1000 35.14%0.6738 1.0000 10.00%

1.0000 1.0000 1.00001.000032 10 0.0000 0.00%1.0000 1.0000 0.00%

1.0000 1.0000 1.00001.000056 10 0.0000 0.00%1.0000 1.0000 0.00%

1.0000 1.0000 1.00001.0000100 10 0.0000 0.00%1.0000 1.0000 0.00%