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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October 2019 to 23 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Test water samples were collected from each batch solution after filtration at the beginning of each renewal (new solutions).
- At the end of the renewal period at 24 and 48 hours (± 1 hour), test water samples were collected from two of four replicate test chambers to measure concentrations of the test substance.
- The samples (50 mL) were collected from mid-depth, placed in 50 mL plastic centrifuge tubes.
- Prior to sample collection, the glassware was rinsed with hot water, followed by Nanopure water, and then dilution water.
- The plastic centrifuge tubes and filters were rinsed with Nanopure water.
Vehicle:
no
Details on test solutions:
DILUTION WATER
- The water used for organism holding and testing was freshwater obtained from a well approximately 40 metres deep located on the EAG Laboratories-Easton site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank where the water was aerated with spray nozzles.
- Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through ultraviolet (UV) steriliser.
- The well water is characterised as moderately-hard water. The specific conductance, hardness,
alkalinity, pH and total organic carbon (TOC) content of the well water during the approximate four-week period immediately preceding the test are presented in Appendix 3 (attached).
- Results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water are presented in Appendix 4 (attached).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- The cladoceran, Daphnia magna, was selected as the test species for this study. This species is representative of an important group of aquatic invertebrates and was selected for use in the test based upon past history of use in the laboratory. Daphnids used in the test were < 24 hours old neonates obtained from cultures maintained by EAG Laboratories, Easton, Maryland. Identification of the species was verified by the supplier of the original stock culture.
- Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test.
- During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.5 to 20.4ºC, the pH of the water ranged from 8.0 to 8.5, and the dissolved oxygen concentrations were ≥ 7.6 mg/L (≥ 84 % of air-saturation).
- The 5 adult daphnids used to supply neonates for the test were held for 20 days prior to collection of the juveniles for testing, and had each produced at least two previous broods. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test.
- The adults showed no signs of disease or stress, no ephippia were produced during the holding period, and there was no immobility in the culture stock was during the two-day period prior to test initiation.
- Loading in each test chamber during the test was approximately 40 mL of test solution per daphnid.

FEEDING
- Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow (YCT; see Appendix 6, attached), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Raphidocelis subcaptitata (formerly Selenastrum capricarnutum). The adults were fed during the 24 hour period prior to test initiation, but neonates were not fed during the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
136 mg/L as CaCO3 in dilution water at test initiation (see Table 4, attached)
Test temperature:
20 ± 1 ºC (see Table 3, attached)
pH:
8.0 to 8.4 (see Table 3, attached)
Dissolved oxygen:
≥ 7.6 mg/L and ≥ 84 % of air saturation (see Table 3, attached)
Salinity:
Not applicable
Conductivity:
Specific conductance 323 μS/cm in dilution water at test initiation (see Table 4, attached)
Nominal and measured concentrations:
Nominal loading rates: 0, 0.3, 1.0, 3.0, 10 and 30 mg/L test item
Details on test conditions:
OBJECTIVE OF THE STUDY
- The objective of this study was to determine the acute effects of the test item on the cladoceran,
Daphnia magna during a 48-hour exposure period under static-renewal test conditions.

EXPERIMENTAL DESIGN
- Due to the low aqueous solubility and complex nature of the test item, testing was performed using a water accommodated fraction (WAF). Daphnia were exposed to five WAF loading rates of test item and a negative control (dilution water) for 48 hours under static-renewal conditions. Four replicate test chambers were maintained in each treatment and control group, with 5 daphnids in each test chamber, for a total of 20 daphnids per loading rate. Nominal WAF loading rates were selected in consultation with the Sponsor based on the results of exploratory range-finding toxicity data (see Table 1, attached). For this test, the term loading rate means the total amount of test item added to the dilution water volume to achieve the respective WAF solutions. Nominal WAF loading rates selected were 0.3, 1.0, 3.0, 10 and 30 mg/L test item. Test concentrations were measured in samples of test water collected from the batches of test solution prepared for each treatment and control group at the beginning of the test, at the 24 hour renewal (± 1 hour) and from two of four replicate test chambers at 24 hours (old solutions) and 48 hours (± 1 hour) to determine concentrations of the test substance. Results of the analyses were used to calculate mean measured test concentrations.
- Neonate daphnids < 24-hours old were impartially assigned to exposure chambers at test initiation. Observations of immobility and other signs of toxicity were made approximately 3.5, 24 and 48 hours after test initiation. Cumulative percent immobility observed in the treatment groups was used to determine the median effective loading rate (EL50) values at 24 and 48-hour intervals.

REFERENCE SUBSTANCES
- The analytical standard used to prepare the analytical calibration standards for the study was received from SPEX CertiPrep on 04 February 2019. It was assigned a testing facility identification number 15232 upon receipt and was stored under ambient conditions. The standard, a liquid, was identified as: 1000 μg/mL Phosphorus; Lot number 24-16PY. The standard had an expiration date of 28 February 2020.
- The internal standard used during the analysis of the samples was received from SPEX CertiPrep on 01 May 2019. It was assigned a testing facility identification number 15412 upon receipt and was stored under ambient conditions. The standard, a liquid, was identified as: 1000 μg/mL Scandium; Lot number 24-33SCY. The standard had an expiration date of 30 April 2020.

TEST APPARATUS
- Test chambers were 250 mL glass beakers filled with approximately 200 mL of test water and loosely covered with plastic petri dishes.
- The depth of the test water in a representative chamber on Day 0 was 7.1 cm. At the 24 hour renewal the depth of the test water in a representative chamber was 6.5 cm.
- All test chambers were labelled with the project number, test concentration and replicate designation. The test chambers were impartially positioned by treatment group in a temperature controlled environmental chamber to maintain the desired test temperature throughout the test period.

NON-GLP RANGE-FINDING TEST
- The range-finding static-renewal study was conducted at nominal WAF loading rates of 0.3, 1.0, 3.0, 10, and 30 mg/L for 48 hours.
- The solutions were filtered through a 0.2 aPES filter prior to adding to the test chambers at initiation and 24 hours.
- The percent immobility in the 0.3, 1.0, 3.0, 10, and 30 mg/L treatment groups at test termination was 0, 0, 20, 70, and 100%, respectively.
- The sublethal effect of lethargy was observed at test termination (see Table 1, attached).

PREPARATION OF TEST LOADING RATES
- The test substance was administered to the test organisms in water. This route of administration was selected because it represents the most likely route of exposure to aquatic organisms. Water accommodated fractions (WAF) were prepared for each loading rate (0.3, 1.0, 3.0, 10, and 30 mg/L) individually in 13.2 L Pyrex aspirator bottles with tubulation and spigot approximately 2 to 3 cm from the bottom.
- Prior to preparation the WAF bottles and 250 mL glass beakers were rinsed with hot tap water and then Nanopure water three times. The Teflon-lined stir bars and vacuum filters were rinsed with Nanopure water three times.
- Dilution water was measured (12 L) and added into the labelled WAF bottle. Appropriate amounts of the test material were weighed into small plastic weigh boats. The test material was rinsed with a portion of the dilution water into the appropriate WAF bottle and the weigh boat was added to the WAF bottle. A Teflon-lined stir bar was added and stirred on a magnetic stir plate for approximately 21 hours for the initial batch and 24 hours at the renewal, with a vortex depth of approximately 30% of the test solution height. The negative control solution was prepared in exactly the same manner as the test solution was prepared, but with dilution water only and no test substance.
- At the beginning of the stirring process, the negative control and 0.3 mg/L treatment group appeared clear and colourless, while the solution for the 1.0 through 30 mg/L treatment groups appeared clear and colourless with white test substance in the water column and water surface that increased with concentration.
- At the termination of stirring process, the negative control appeared clear and colourless. The 0.3, 1.0, and 3.0 mg/L test solutions appeared clear and colorless with precipitate in the water column and oil globules on the water surface. The 10 and 30 mg/L solution was clear and colourless with precipitation on the surface and throughout the water column.
- The solutions were allowed to settle for approximately 1.5 hours to allow phase separation before decanting the aqueous phase. At the settling termination, the negative control was clear and colourless. The 0.3, 1.0, and 3.0 mg/L test solutions were clear and colourless with oil globules on the water surface. The appearance of the 10 and 30 mg/L test solutions was clear and colourless with precipitate on the water surface and on the bottom of the WAF bottle.
- The solutions were filtered into a glass secondary container through a vacuum filter with a 0.2 aPES filter. All solutions appeared clear and colourless with no indication of precipitates when viewed under a dissecting microscope. Approximately 200 mL of the filtered test solutions were added to each test replicate.

ENVIRONMENTAL CONDITIONS
- The test system was illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight. The lights were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Inc. Model 840006 light meter.
- The target test temperature during the study was 20 ± 1C. Temperature was measured in each test chamber at the beginning of the test, at approximately 24-hours (old and new solutions) during the test and at the end of the test using a digital thermometer. Water temperature also was monitored continuously in adjacent container of water using a validated environmental monitoring system (PointView Real-time Data Reporting System). The system measurements were calibrated prior to exposure initiation with a digital thermometer.
- Dissolved oxygen and pH were measured in each test chamber at the beginning of the test, at approximately 24-hour intervals (old and new solutions) during the test, and at the end of the test. Dissolved oxygen was measured using a Thermo Scientific Orion Star A213 dissolved oxygen meter, and measurements of pH were made using a Thermo Scientific Orion Dual Star pH/ISE meter.
- Hardness, alkalinity, and specific conductance in the dilution water at the beginning of the test were measured. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using a Thermo Orion Star A122 Portable Conductivity Meter.

PROCEDURES AND BIOLOGICAL OBSERVATIONS
- Prior to test initiation, neonate daphnids < 24 hours old were collected from the cultures and were indiscriminately distributed one to two at a time to the transfer containers until each contained its compliment of 10 daphnids.
- To initiate the test, the transfer containers were impartially assigned to test chambers, and the daphnids were released into the test chambers. All daphnid transfers were conducted below the water surface using wide-bore pipettes.
- All organisms were observed periodically during the test to determine the number of immobile organisms in each treatment group. Those daphnids that are not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilised (even if they can still move their antennae).
- The numbers of individuals exhibiting signs of toxicity or abnormal behaviour also were evaluated.
- Observations were made approximately 3.5, 24 and 48 hours (± 1 hour) after test initiation.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.56 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence interval 0.36 to 0.75 mg/L
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL100
Effect conc.:
2.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
0.36 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits 1.0 to 3.0 mg/L
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL100
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Details on results:
MEASUREMENT OF TEST LOADING RATES
- Nominal WAF loading rates selected for use in the study were 0.3, 1.0, 3.0, 10, and 30 mg/L test item. Test solutions in the test chambers at these nominal loading rates appeared clear and colourless during the test, with no evidence of precipitation observed and when viewed under a microscope.
- Results of analyses to measure concentrations of test item in the test solutions samples collected
during the test are presented in Table 2 (attached).
- The mean measured loading rate was < LOD in the 0.3 mg/L test item treatment group due to the difficult nature of the test material and analytical method limitations.
- Over the course of the study, percent recoveries ranged from < LOD to 134% of nominal WAF loading rates. Measured loading rates in the remaining treatment group means ranged from 24 to 52 % of nominal. When measured loading rates of the samples collected during the test were averaged, the mean measured test loading rates for this study were < LOD, 0.36 0.75, 2.4, and 16 mg/L.
- For results > LOD, percent recoveries of nominal WAF loading rates were 36, 25, 24, and 52%, respectively. The results of the study were based on the mean measured loading rates, and results are also presented based on nominal WAF loading rates.

OBSERVATIONS AND MEASUREMENTS
- Measurements of temperature, dissolved oxygen and pH of the water in the test chambers are presented in Table 3 (attached).
- Water temperatures were within the 20 ± 1 ºC range established for the test (see Table 3, attached).
- Dissolved oxygen concentrations remained ≥ 7.6 mg/L (≥ 84 % of air saturation) throughout the test (see Table 3, attached).
- Measurements of pH ranged from 8.0 to 8.4 during the test (see Table 3, attached).
- The measurements of hardness, alkalinity, and specific conductance in the dilution water at test initiation were typical of Eurofins-Easton well water (see Table 4, attached).
- Light intensity at test initiation was 560 lux at the surface of the water of one representative test chamber.
- Daily observations of immobility and other signs of toxicity observed during the test are presented in Table 5 (attached). All daphnids in the negative control group appeared normal throughout the test. All daphnids in the 0.3 and 1.0 mg/L nominal WAF loading rates (< LOD mean measured and 0.36 mg/L mean measured) also appeared normal throughout the test with no immobility or overt signs of toxicity noted. The percent immobility in the 3.0, 10, and 30 mg/L nominal WAF loading rates (0.75, 2.4, and 16 mg/L mean measured) was 90, 100, and 100% respectively at 48 hours. Lethargy prior to immobilisation was observed in the 10 and 30 mg/L nominal WAF loading rates (2.4 and 16 mg/L mean measured). The EL50 value at 48 hours was determined from the immobility data and is shown in Table 6 (attached).

VALIDITY OF THE TEST
- The following criteria were used to judge the validity of the test:
(i) Immobility of the daphnids in the control group will not exceed 10 % by the end of the test. In this study, no immobility was observed in the control group.
(ii) The dissolved oxygen concentration will be ≥3 mg/L throughout the test. In this study, the dissolved oxygen concentration remained ≥ 7.6 mg/L (≥ 84 % of air saturation).
(iii) Evidence should be available to demonstrate that the concentrations of the test substance in solution have been satisfactorily maintained within ± 20 % of the nominal test concentrations. In this study, measured concentrations were not maintained within ± 20 % of the nominal WAF loading rates due to the difficult nature of the test material and difficulty in processing of the test solutions, and ranged from 24 to 52 % of nominal test concentrations.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
DATA ANALYSES
- The immobility data were analysed using the computer program of C. E. Stephan. The program was designed to calculate the EL50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation.
- Based on the immobility pattern in the study, nonlinear interpolation was used to calculate the 48 hour EL50 value and to calculate the 95 % confidence intervals for the nominal WAF loading rates and mean measured loading rates.
- Since there was < 50 % immobility at 24 hours, the 24-hour EL50 value was estimated to be greater than the highest concentration tested. Due to the method used to calculate the 48-hour EL50 value, the slope of the concentration-response curve could not be calculated. Based on the immobility pattern, the NOELR was determined by visual interpretation of the data.
Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured loading rates, the 48-hour EL50 was determined to be 0.56 mg/L. The concentration causing no immobility at test end was 0.36 mg/L and the concentration causing 100 % immobility was 2.4 mg/L. The NOELR was 0.36 mg/L. Based on nominal WAF loading rates and immobility data, the 48-hour EL50 determined to be 2.0 mg/L with a 95% confidence interval of 1.0 to 3.0 mg/L. The highest loading rate causing no immobility at test end was 1.0 mg/L, the lowest loading rate causing 100% immobility at test end was 10 mg/L, and the no-observed effect loading rate was 1.0 mg/L.
Executive summary:

GUIDELINE

The study protocol was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test; U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OCSPP Number 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids; and ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, the investigation was performed using a water accommodated fraction (WAF). Daphnia were exposed to five nominal WAF loading rates of 0.3, 1.0, 3.0, 10 and 30 mg test item and a control (dilution water) for 48 hours under static renewal conditions. Test concentrations were measured in samples of test water collected from the batches of test solution prepared for each treatment and control group at the beginning of the test, at the 24-hour renewal (± 1 hour) and from two of four replicate test chambers at 24 hours (old solutions) and 48 hours (± 1 hour) to determine concentrations of the test substance. Results of the analyses were used to calculate mean measured test concentrations.

 

RESULTS

The mean measured loading rate was < LOD in the 0.3 mg/L test item treatment group due to the difficult nature of the test material and analytical method limitations. Over the course of the study, percent recoveries ranged from < LOD to 134% of nominal WAF loading rates. Measured loading rates in the remaining treatment group means ranged from 24 to 52 % of nominal. When measured loading rates of the samples collected during the test were averaged, the mean measured test loading rates for this study were < LOD, 0.36 0.75, 2.4, and 16 mg/L. For results > LOD, percent recoveries of nominal WAF loading rates were 36, 25, 24, and 52%, respectively.

 

CONCLUSION

Based on mean measured loading rates, the 48-hour EL50 was determined to be 0.56 mg/L. The concentration causing no immobility at test end was 0.36 mg/L and the concentration causing 100 % immobility was 2.4 mg/L. The NOELR was 0.36 mg/L. Based on nominal WAF loading rates and immobility data, the 48-hour EL50 determined to be 2.0 mg/L with a 95% confidence interval of 1.0 to 3.0 mg/L. The highest loading rate causing no immobility at test end was 1.0 mg/L, the lowest loading rate causing 100% immobility at test end was 10 mg/L, and the no-observed effect loading rate was 1.0 mg/L.

Description of key information

Based on mean measured loading rates, the EL50 (48 h) value for Daphnia magna was determined to be 0.56 mg/L (OECD 202 and OCSPP 850.1010).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2 mg/L

Additional information

GUIDELINE

The study protocol was based on procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test; U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OCSPP Number 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids; and ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, the investigation was performed using a water accommodated fraction (WAF). Daphnia were exposed to five nominal WAF loading rates of 0.3, 1.0, 3.0, 10 and 30 mg test item and a negative control (dilution water) for 48 hours under static-renewal conditions. Test concentrations were measured in samples of test water collected from the batches of test solution prepared for each treatment and control group at the beginning of the test, at the 24-hour renewal (± 1 hour) and from two of four replicate test chambers at 24 hours (old solutions) and 48 hours (± 1 hour) to determine concentrations of the test substance. Results of the analyses were used to calculate mean measured test concentrations.

 

RESULTS

The mean measured loading rate was < LOD in the 0.3 mg/L test item treatment group due to the difficult nature of the test material and analytical method limitations. Over the course of the study, percent recoveries ranged from < LOD to 134% of nominal WAF loading rates. Measured loading rates in the remaining treatment group means ranged from 24 to 52 % of nominal. When measured loading rates of the samples collected during the test were averaged, the mean measured test loading rates for this study were < LOD, 0.36 0.75, 2.4, and 16 mg/L. For results > LOD, percent recoveries of nominal WAF loading rates were 36, 25, 24, and 52%, respectively.

 

CONCLUSION

Based on mean measured loading rates, the 48-hour EL50 was determined to be 0.56 mg/L. The concentration causing no immobility at test end was 0.36 mg/L and the concentration causing 100 % immobility was 2.4 mg/L. The NOELR was 0.36 mg/L. Based on nominal WAF loading rates and immobility data, the 48-hour EL50 determined to be 2.0 mg/L with a 95% confidence interval of 1.0 to 3.0 mg/L. The highest loading rate causing no immobility at test end was 1.0 mg/L, the lowest loading rate causing 100% immobility at test end was 10 mg/L, and the no-observed effect loading rate was 1.0 mg/L.