2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Sep 2019 to 17 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Test water samples were collected at the beginning (new solutions) and end (old solutions) of each renewal period of the test to determine concentrations of the test substance.
- Test water samples were collected from each batch solution after filtration at the beginning of the test and at each renewal (new) at 24, 48, and 72 hours (± 1 hour), with the exception at 48 and 72 hours which were sampled slightly outside the ± 1 hour range.
- At the end of the renewal period at 24, 48, 72, and 96 hours (± 1 hour), test water samples were collected from each test chamber (old solutions) to measure concentrations of the test substance. The samples (50 mL) were collected from mid-depth, placed in 50 mL plastic centrifuge tubes, and stabilised for later analysis.
- Sample collection from the 100 mg/L treatment group was discontinued after 48 hours due to 100 % mortality.
- Prior to sample collection, the glassware was rinsed with hot water, followed by Nanopure 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 Eurofins-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 in the test system, the water was filtered to 0.45 µm to remove fine particles and was passed through an 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). The 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):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- The fathead minnow (Pimephales promelas) was selected as the test species for this study. This species is representative of an important group of aquatic vertebrates and was selected for use in the test based upon past history of use in the laboratory. Fathead minnows used in the test were received as juveniles from Osage Catfisheries Inc, Missouri. Identification of the species was verified by the supplier.
- All fish used in the test were juveniles from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest. The average total length of the 10 negative control fish measured at the end of the test was 3.0 cm, with a range of 2.5 to 3.6 cm. The average wet weight (blotted dry) of the 10 negative control fish measured at the end of the test was 0.18 grams, with a range of 0.07 to 0.33 grams. Loading was defined as the total wet weight of fish per litre of test solution, and was 0.18 g fish/L.
- The fish were held for at least 14 days prior to the test 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 22.2 to 23.5 °C, the pH of the water ranged from 8.2 to 8.6, and the dissolved oxygen concentrations were ≥ 8.0 mg/L (≥ 92 % of saturation). During this 2-week period, the fish in the lot used for the test showed no signs of disease or stress and there were no mortalities.

FEEDING
- Daily during the holding period, except for periods of fasting prior to testing, the fish were fed brine shrimp nauplii (Artemia sp.) supplied by Brine Shrimp Direct, Ogden, Utah. The fish were not fed for two days prior to the test or during the test.
- The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the feed are presented in Appendix 5 (attached).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

132 mg/L as CaCO3 in dilution water at test initiation (see Tabel 4, attached)

Test temperature:

22 ± 1 °C (see Table 3, attached)

pH:

7.9 to 8.5 (see Table 3, attached)

Dissolved oxygen:

≥ 7.7 mg/L and ≥ 89 % of saturation (see Table 3, attached)

Salinity:

Not applicable

Conductivity:

Specific conductance 302 μS/cm on Day 0 (see Table 4, attached)

Nominal and measured concentrations:

Nominal loading rates of 1.0, 3.0, 10, 30 and 100 mg/L test item

Details on 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). Fathead minnows were exposed to five WAF loading rates of 1.0, 3.0, 10, 30, and 100 mg/L test item plus a negative control (dilution water) for 96 hours under static renewal conditions.
- Two replicate test chambers were maintained in each treatment and control group, with five fish in each test chamber, for a total of 10 fish per concentration. Nominal WAF loading rates were selected in consultation with the Sponsor based on the results of exploratory Non-GLP range-finding toxicity data (see Table 1, attached).
- For this test, the term loading rate means the total amount of test substance added to the dilution water volume to achieve the respective WAF solutions. Test concentrations were measured in samples of test water collected from each treatment and control group at the beginning, at each 24 hour interval, and at test termination. Results of the analyses were used to calculate mean measured test concentrations. Results of the study are based on mean measured loading rates.
- Juvenile fathead minnows were impartially assigned to exposure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 4.5, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to determine LL50 values at 24, 48, 72 and 96 hours. The no-observed-effect loading rate (NOELR) was determined by visual analysis of the mortality data.

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; CAS number H2O[7732-18-5]. 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; CAS number HNO3[7697-37-2]. The standard had an expiration date of 30 April 2020.

TEST APPARATUS
- Test chambers were 25 L stainless steel aquaria containing approximately 5 L of test solution. The depth of the test water in a representative chamber was 6.0 cm.
- Each test chamber was labelled with the study number, test concentration and replicate designation.
- Test chambers were impartially positioned in two temperature controlled water baths set to maintain the desired test temperature throughout the exposure period with one replicate of each treatment group and control split between the two units.

NON-GLP RANGE-FINDING TEST
- Two range-finding studies were conducted at nominal WAF loading rates of 1.0, 3.0, 10, 30, and 100 mg/L for 96 hours.
- The first static test resulted in percent mortalities of 0, 0, 0, 0, and 40 % in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups at test termination respectively. Sublethal effects at test termination included lethargy, loss of equilibrium, and lying on the bottom of the tank (see Table 1, attached).
- A second range finding test was conducted using a static design with daily renewals with the same nominal WAF loading rates. The solutions were filtered through a 0.2 aPES filter prior to adding to the test chambers. Percent mortality in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups at test termination was 0, 0, 0, 0, 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 (1.0, 3.0, 10, 30, and 100 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 were rinsed with hot tap water and then Nanopure water. The Teflon-lined stir bars and vacuum filters were rinsed with Nanopure water.
- 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 24 hours, 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 appeared clear and colourless, while the solution for the 1.0 through 100 mg/L treatment groups appeared clear colourless with white test substance in the water column and water surface that increased with concentration.
- At the termination of the stirring process, the negative control appeared clear and colourless. The 1.0 mg/L test solution appeared clear and colorless with white precipitate in the water column and an oil slick on the water surface. The 3.0 mg/L solution was clear and colourless with white precipitation on the surface and throughout the water column, and with large brown precipitate on the surface with an oil slick. The 10 and 30 mg/L solutions were clear and colourless with large brown chunks and small white precipitate on the water surface, as well as in the water column. The brown precipitate observed at the termination of the stirring was observed once more in the 30 mg/L test solutions at the Day 1 preparation but was not observed in subsequent preparations. The 100 mg/L was clear and colourless with white precipitate on the surface and throughout the water column.
- The solutions were allowed to settle for approximately 1 hour to allow phase separation before decanting the aqueous phase. At the settling termination, the negative control was clear and colourless. The 1.0 mg/L test solution was clear and colourless with white precipitate on the bottom of the vessel. The appearance of the test solutions for the 3.0 through 100 mg/L treatment groups were clear and colourless with white precipitate on the sides and bottom of the vessel and on the surface of the solution. The solutions were filtered into a glass secondary container through a vacuum filter with a 0.2 aPES filter. All solutions appeared clear and colorless with no indication of precipitates when viewed under a dissecting microscope. Five litres of the filtered test solutions were added to each test replicate.

ENVIRONMENTAL CONDITIONS
- The test systems were 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 Model 840006 light meter.
- The test was conducted at a target water temperature of 22 ± 1 °C. Temperature was measured in each test chamber at the beginning of the test, at approximately 24-hour intervals prior to and after renewals during the test, and at the end of the test using a digital thermometer. Temperature also was monitored continuously in the negative control test chamber of each water bath 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 batch solution at the beginning of each renewal period and in each test chamber at approximately 24-hour intervals during the test, and at the end of the test. Dissolved oxygen was measured using a Thermo Scientific Orion Star A213 Benchtop RDO/DO meter, and measurements of pH were made using a Thermo Scientific Orion DUAL STAR pH/ISE meter. When 100% mortality occurred in a test chamber, measurements of temperature, dissolved oxygen and pH were taken in that test chamber and then discontinued.
- Hardness, alkalinity and specific conductance in the dilution water were measured at the beginning of the test. Hardness and alkalinity measurements were made by titration based on methods in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using a Thermo Scientific Orion Star A122 portable conductivity meter.

PROCEDURES AND BIOLOGICAL OBSERVATIONS
- Prior to test initiation, juvenile fathead minnows were collected from the holding tank. To initiate the test, the fish were impartially distributed one or two at a time to the test chambers until each contained five fish.
- All organisms were observed periodically during the test to determine the number of mortalities in each treatment group. Mortality was defined as the lack of visible movement (e.g., lack of fin or opercular movement) and the lack of response to gentle prodding in the fish. Dead fish were removed from the test chambers at each observation interval.
- The numbers of individuals exhibiting signs of toxicity or abnormal behaviour also were evaluated. Observations were made approximately 4.5, 24, 48, 72 and 96 hours after test initiation.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence limit 8.7 to 31 mg/L

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

31 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % confidence limit 30 to 100 mg/L

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mortality (fish)

Details on results:

MEASUREMENT OF TEST LOADING RATES
- Nominal WAF loading rates selected for use in this study were 1.0, 3.0, 10, 30, and 100 mg/L test item. Test solutions in the test chambers at these nominal concentrations appeared clear and colourless during the test, with no evidence of precipitation observed, and also when viewed under a microscope.
- Results of analyses to measure concentrations of test item, in the test solution samples collected during the test are presented in Table 2. Measured loading rates of the samples ranged from approximately < LOD to 69 % of nominal. When measured concentrations of the samples were averaged, the mean measured loading rates for this study were < LOD, 1.0, 4.1, 8.7, and 31 mg/L, representing 0, 33, 41, 29, and 31 % of nominal concentrations, respectively. The results of the study were based on the mean measured loading rates, with results based on nominal WAF loading rates also reported.

OBSERVATIONS AND MEASUREMENTS
- Measurements of temperature, pH and dissolved oxygen of the water in the test chambers are presented in Table 3 (attached).
- Water temperatures were within the 22 ± 1 °C range established for the test.
- Measurements of pH ranged from 7.9 to 8.5 during the test.
- Dissolved oxygen concentrations remained ≥ 7.7 mg/L (≥ 89 % of saturation) throughout the test.
- The measurements of hardness, alkalinity and specific conductance in the dilution water at the beginning of the test were typical of Eurofins-Easton well water (see Table 4, attached).
- Light intensity at the beginning of the test was 983 lux at the surface of the water of one representative test chamber.
- Daily observations of mortality and other signs of toxicity observed during the test are presented in Table 5 (attached).
- All fathead minnows in the negative control group appeared normal throughout the test.
- All fish in the 1.0 and 3.0 mg/L nominal WAF loading rate (< LOD and 1.0 mg/L mean measured) treatment groups also appeared normal throughout the test, with no mortalities or overt signs of toxicity observed.
- Percent mortality in the 10, 30, and 100 mg/L nominal WAF loading rate (4.1, 8.7, and 31 mg/L mean measured) treatment groups at test termination was 0, 10, and 100 %, respectively. Signs of toxicity observed among the fish in the 10, 30, and 100 mg/L nominal WAF loading rate (4.1, 8.7, and 31 mg/L mean measured) treatment groups during the course of the study included loss of equilibrium, surfacing, lethargy, and lying on the bottom of the test chamber.
- Therefore, in this study, the no-mortality loading rate was 10 mg/L (4.1 mg/L mean measured), the 100% mortality loading rate was 100 mg/L (31 mg/L mean measured), and the NOELR was 3.0 mg/L nominal WAF loading rate (1.0 mg/L mean measured). The LL50 values at 24, 48, 72 and 96 hours were determined from the mortality data and are shown in Table 6 (attached).

VALIDITY OF THE TEST
- The following criteria were used to judge the validity of the test and were met in this study with one exception:
i) Mortality and/or signs of disease or stress in the fish in the control group will not exceed 10 % (or one fish if less than 10 are used) by the end of the test. In this study, there were no mortalities, or signs of disease or stress observed in the control group.
ii) The dissolved oxygen concentration will be at least 60% of the air-saturation value throughout the test. In this study, the dissolved oxygen concentration remained ≥ 89 % of 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, due to the difficulty of the processing of the WAF solutions, measured concentrations was < LOD for the 1.0 mg/L nominal WAF loading rate due to the difficult nature of the test material and difficulty in processing of the test solutions, and ranged from 29 to 41 % of nominal test concentrations. The measured concentrations for the 3.0 through 100 mg/L nominal WAF loading rates ranged from 18 to 56 % of nominal test concentrations.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

DATA ANALYSES
- The mortality data were analySed using the computer program of C. E. Stephan. The program was designed to calculate the LL50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation. Based on the mortality pattern in this study, nonlinear interpolation was used to calculate the 24, 48, 72, and 96-hour LL50 values and to calculate the 95 % confidence intervals.
- Due to the method used to calculate the 96-hour LL50 value, the slope of the concentration response curve could not be calculated. As the lowest treatment group’s measured recovery was < LOD, a statistical analysis of the NOELR was precluded. Therefore, the no-observed-effect loading rate (NOELR) determined by visual interpretation of the data.

Validity criteria fulfilled:

yes

Conclusions:

Based on the mean measured concentrations, the 96-hour LL50 value was 14 mg/L, with a 95% confidence interval of 8.7 to 31 mg/L. The no-mortality loading rate was 4.1 mg/L, the 100% mortality loading rate was 31 mg/L and the NOELR was 1.0 mg/L. Based on the nominal WAF loading rates, the 96-hour LL50 value was 48 mg/L, with a 95% confidence interval of 30 to 100 mg/L. The no-mortality loading rate was 10 mg/L, the 100% mortality loading rate was 100 mg/L and the NOELR was 3.0 mg/L.

Executive summary:

GUIDELINE

The study protocol was based on was procedures in the OECD Guidelines for Testing of Chemicals, Guideline 203: Fish, Acute Toxicity Test; the U.S. EPA Series 850 - Ecological Effects Test Guidelines, OCSPP 850.1075: Freshwater and Saltwater Fish Acute Toxicity Test; and ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.

 

METHODS

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static conditions to five WAF loading rates of 1.0, 3.0, 10, 30 and 100 mg/L plus a negative control consisting of dilution water.

 

RESULTS

Test solutions in the test chambers at these nominal concentrations appeared clear and colourless during the test, with no evidence of precipitation observed, and also when viewed under a microscope. Measured loading rates of the samples ranged from approximately < LOD to 69 % of nominal. When measured concentrations of the samples were averaged, the mean measured loading rates for the study were < LOD, 1.0, 4.1, 8.7, and 31 mg/L, representing 0, 33, 41, 29, and 31 % of nominal concentrations, respectively.

 

CONCLUSION

Based on the mean measured concentrations, the 96-hour LL50 value was 14 mg/L, with a 95% confidence interval of 8.7 to 31 mg/L. The no-mortality loading rate was 4.1 mg/L, the 100% mortality loading rate was 31 mg/L and the NOELR was 1.0 mg/L. Based on the nominal WAF loading rates, the 96-hour LL50 value was 48 mg/L, with a 95% confidence interval of 30 to 100 mg/L. The no-mortality loading rate was 10 mg/L, the 100% mortality loading rate was 100 mg/L and the NOELR was 3.0 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

48 mg/L

Additional information

GUIDELINE

The study protocol was based on was procedures in the OECD Guidelines for Testing of Chemicals, Guideline 203: Fish, Acute Toxicity Test; the U.S. EPA Series 850 - Ecological Effects Test Guidelines, OCSPP 850.1075: Freshwater and Saltwater Fish Acute Toxicity Test; and ASTM Standard E 729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians.

 

METHODS

Fathead minnows (Pimephales promelas) were exposed for 96 hours under static conditions to five WAF loading rates of 1.0, 3.0, 10, 30 and 100 mg/L plus a negative control consisting of dilution water.

 

RESULTS

Test solutions in the test chambers at these nominal concentrations appeared clear and colourless during the test, with no evidence of precipitation observed, and also when viewed under a microscope. Measured loading rates of the samples ranged from approximately < LOD to 69 % of nominal. When measured concentrations of the samples were averaged, the mean measured loading rates for the study were < LOD, 1.0, 4.1, 8.7, and 31 mg/L, representing 0, 33, 41, 29, and 31 % of nominal concentrations, respectively.

 

CONCLUSION

Based on the mean measured concentrations, the 96-hour LL50 value was 14 mg/L, with a 95% confidence interval of 8.7 to 31 mg/L. The no-mortality loading rate was 4.1 mg/L, the 100% mortality loading rate was 31 mg/L and the NOELR was 1.0 mg/L. Based on the nominal WAF loading rates, the 96-hour LL50 value was 48 mg/L, with a 95% confidence interval of 30 to 100 mg/L. The no-mortality loading rate was 10 mg/L, the 100% mortality loading rate was 100 mg/L and the NOELR was 3.0 mg/L.