2-Propenoic acid, methyl ester, reaction products with mixed O,O-bis(branched and linear pentyl and iso-Bu) phosphorodithioates

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-19 to 2012-05-18

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Range-finding studyStability - A sample of each loading rate WAF was taken for analysis at 0 and 48 hours. All samples were stored at approximately -20°C prior to analysis. Only concentrations within the range to be used for the definitive test were analysed.Definitive testWater samples taken from control and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis. All samples were stored at approximately -20°C prior to analysis.Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.Samples at the No Observed Effect Loading Rate and above only were analysed.

Vehicle:

no

Details on test solutions:

Validation of mixing periodPre-study investigational work was performed to determine if stirring for a prolonged period produced significantly higher levels of total organic carbon (TOC), as an indicator of soluble organic substances, inthe WAF.A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for 95 hours. After a 1-hour standing period, the mixtures were removed by siphon and samples taken for TOC.Range-finderAmounts of test item (10, 40 and 200 mg) were each separately added to the surface of 10, 4 and 2 litres of dechlorinated tap water to give 0.1, 10 and 100 mg/L loading rates respectively. After the addition of the test item the water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations of made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was justified to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 - 100 mL discarded) to give the 0.1, 10 and 100 mg/L loading rate WAFs. Microscopic observations were performed onthe WAFs after filtering and showed no micro-particles of the test item present.Definitive testAmounts of test item (100, 180, 128, 140 and 250 mg) were each separately added to the surface of 10, 10, 4, 2.5 and 2.5 litres of dechlorinated tap water to give 10, 18, 32, 56 and 100 mg/L loading rates respectively.The test concentrations were prepared in the same manner as for the range finder.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM- Common name: Daphnia magna- Source: in-house laboratory culture, Harlan Laboratories.- Age at study initiation (mean and range, SD): < 24 hours- Breeding: parthogenesis- Feeding during test: NoACCLIMATION- Acclimation period: not applicable culture maintained in-house. Adult Daphnia maintained in 150 mL glass beakers containing Elendt M7 medium. Gravid adults isolated day before initiation of test so young daphnids produced overnight were less than 24 hours old.- Acclimation conditions (same as test or not): culture conditions same as test.- Type and amount of food: Tetramin flake food suspension and algal suspension (Desmodesmus subspicatus)- Feeding frequency: daily- Health during acclimation (any mortality observed): not applicable culture maintained in-house. Adult Daphnia maintained in 150 mL glass beakers containing Elendt M7 medium. Gravid adults isolated day before initiation of test so young daphnids produced overnight were less than 24 hours old.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

250 mg/L

Test temperature:

21 - 22°C

pH:

7.9 - 8.1

Dissolved oxygen:

8.4 - 8.9 mg O2/L

Salinity:

Not measured

Nominal and measured concentrations:

Definitive testnominal: 10, 18, 32, 56 and 100 mg/L loading rate WAF

Details on test conditions:

TEST SYSTEM- Test vessel: 250 mL glass jar- Type (delete if not applicable): closed- Material, size, headspace, fill volume: 200 mL test preparation- Aeration: no- No. of organisms per vessel: 10- No. of vessels per concentration (replicates): 2- No. of vessels per control (replicates): 2TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: reconstituted water- Conductivity: < 5 µS/cm- Culture medium different from test medium: noOTHER TEST CONDITIONS- Adjustment of pH: In reconstituted water only- Photoperiod: 16 hours light, 8 hours dark with a 20 minute dawn and dusk transition periods- Light intensity: 632 - 750 luxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :Immobilisation or adverse reaction: 24 and 48 hours after start of exposureWater temperature: dailyDissolved oxygen content and pH: start and termination of testTEST CONCENTRATIONS- Range finding study- Test concentrations: 1.0, 10 and 100 mg/L loading rate WAF- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate at test concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI = 36 - 47 mg/L loading rate WAF

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate WAF

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: loading rate WAF

Details on results:

Range finding:No immobilisation was observed at 1.0 and 10 mg/L loading rate WAF. However, immobilisation was observed at 100 mg/L loading rate WAF.Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L laoding rate WAF were chosen for the definitive test.Chemical analysis of the 10 mg/L loading rate WAF at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, i.e. 0.34 mg/L. Chemical analysis of the 100 mg/L loading rate WAF showed measured concentrations of 0.577 and 0.440 mg/L respectively.Definitive test:Inspection of the mobilisation data at 24 hours and analysis of the immobilisation data by the probit method at 48 hours based on the nominal loading rates gave the following results:EL50 (24h) > 100 mg/L loading rate WAFEL50 (48 h) = 41 mg/L loading rate WAF (95% CI = 36 - 47 mg/L)The Lowest Effect Loading rate was considered to be 32 mg/L loading rate WAF. The No Observed Effect Loading rate after 24 and 48 hours exposure were 100 and 18 mg/L loading rate WAF respectively.The slope and its standard error of the response curve at 48 hours was 9.6 (SE = 2.0)Physicochemical measurementsValidation of mixing period: There was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours.Temperature was maintained at 21 - 22°C throughout the test. There were no treatment related differences for oxygen concentration or pH.The oxygen concentration in some test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.At the start and end of the mixing period the 10 mg/L loading rate was observed to be a clear colourless water column with an oily slick of test item at the water surface whilst the 18, 32, 56 and 100 mg/L loading rates were observed to be clear colourless water columns with an oily slick of test item floating at the surface and oily globules dispersed throughout the water column and settled at the bottom of the vessel. After 23 hours stirring and a 1-hour standing period the 10 mg/L loading rate was observed to be a clear colourless water column with an oily globules of test item floating at the surface whilst the 18, 32, 56 and 100 mg/L loading rates were observed to be clear colourless water columns with an oily slick of test item floating at the surface and oily globules dispersed throughout the water column and settled at the bottom of the vessel. Visual examination of the WAF showed no microdispersions or undissolved test item to be present. After siphoning and for the duration of the test the 8, 32, 56 and 100 mg/L loading rates observed to be a clear colourless solutions.Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, i.e. 0.34 mg/L, to 1.02 mg/L. Chemical analysis of the test preparations at 48 hours showed measured test concentrations to range from less than LOQ to 0.806 mg/L. Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the dissolved test item was close to or below the quantifiable limit of the analytical method , the results were based on nominal loading rates only.

Results with reference substance (positive control):

- Results with reference substance valid? Yes- Mortality:- EC50/LC50: (48h) = 1.1 mg/L (95% CI = 1.0 - 1.3 mg/L)- NOEC (48h) = 0.56 mg/L

See attached document

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to Daphnia magna gave a 48 hour EL 50 value of 41 mg/L loading rate WAF with 95% confidence limits of 36 - 47 mg/L loading rate WAF. The No Observed Effect Loading rate at 48 hours was 18 mg/L loading rate WAF.

Executive summary:

Test Guidance

OECD Guideline No 202 and EC Method C.2

Method

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21 - 22°C under static test conditions. The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

The acute toxicity of the test item to Daphnia magna gave a 48 hour EL 50 value of 41 mg/L loading rate WAF with 95% confidence limits of 36 - 47 mg/L loading rate WAF. The No Observed Effect Loading rate at 48 hours was 18 mg/L loading rate WAF. The Lowest Effect Loading rate was considered to be 32 mg/L loading rate WAF.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, i.e. 0.34 mg/L, to 1.02 mg/L. Chemical analysis of the test preparations at 48 hours showed measured test concentrations to range from less than LOQ to 0.806 mg/L.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the dissolved test item was close to or below the quantifiable limit of the analytical method , the results were based on nominal loading rates only.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001-05-28 to 2002-06-08

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable.

Vehicle:

no

Details on test solutions:

.TEST WATER- Reconstituted water used for both the range-finding and definitive test is defined in Appendix 1 (attached).RANGE-FINDING TEST- The loading rate to be used in the definitive test was determined by a preliminary range-finding test.- In the range-finding test, Daphnia magna were exposed to nominal loading rates of 10 amd 100 mg/L. The test material was prepared as a WAF.- Amounts of test material (20 and 200 mg) were separately added to the surface of 2 L of reconstituted water to give the 10 and 100 mg/L loading rates respectively.- After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface.- Stirring was stopped after 24 hours and the mixtures were allowed to stand for one hour.- A wide bore glass tube, covered at one end with Parafilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel.- A length of Tygon tubing was inserted into the glass tube and pushed through the Parafilm seal.- The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs.- Microscopic inspection of the WAFs showed no microdispersions or undissolved test material, therefore a glass wool plug was not used to filter the WAFs.

Test organisms (species):

Daphnia magna

Details on test organisms:

- The test was carried out using 1st instar Daphnia magna dervied from in-house laboratory cultures.- Adult Daphnia were maintained in polypropylene vessels containing approximately 2 L of reconstituted water in a temperature controlled room at 21 °C.- The lighting cycle was controlled to give 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.- Each culture was fed daily with a suspension of algae (Chlorella sp.).- Culture conditions ensured that reproduction was by parthenogenesis.- Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.- These young were removed from the cultures and used for testing.- The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

250 mg/L total theoretical hardness as CaCO3

Test temperature:

21 °C (see Appendix 2, attached)

pH:

pH 8.0 - 8.6 (see Appendix 2, attached)

Dissolved oxygen:

8.5 to 8.7 mg O2/L (see Appendix 2, attached)

Salinity:

Not applicable

Nominal and measured concentrations:

RANGE-FINDING TEST- Nominal concentrations of 10 and 100 mg/LDEFINITIVE TEST- Nominal concentration of 100mg/L

Details on test conditions:

RANGE-FINDING TEST- Daphnids (10) were placed in each test and control vessel and maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods.- Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation.- After 24 and 48 hours, the number of immobilised Daphnia magna were recorded.- The control group was maintained under identical conditions but not exposed to the test item.- Data from preparation of the control medium was shared with similar concurrent studies.DEFINITIVE TEST- Based on the results of the range-finding test, a 'limit test' was conducted at a single loading rate of 100 mg/L to confirm that no immobilisation or adverse reactions to exposure were observed.- As in the range-finding test, 250 mL glass jars containing approximately 200 mL of test preparation were used.- At the start of the test daphnids (10) were placed in each test and control vessel at random.- Four replicate test vessels were prepared with duplicate controls.- The test vessels were the covered to reduce evaporation and maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.- Daphnids were not individually identified, received no food during exposure, and the test vessels were not aerated.- The control group was maintained under identical conditions but not exposed to the test item.- Data from preparation of the control medium was shared with similar concurrent studies.- Test preparations were not renewed during the exposure period.- Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST- Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1 (attached).- No immobilisation was observed at the 10 and 100 mg/L loading rate WAFs- Based on this information, a single loading rate of four replicates of 100 mg/L with a stirring period of 24 hours followed by a one hour standing period, was selected for the definitive test.- The experimental design conforms to a 'limit test' to confirm that no immobilisation or adverse reactions to exposure were observed.DEFINITIVE TEST- Cumulative immobilisation data from the exposure of Daphnia magna to the test material are given in Table 2 (attached).- There was no immobilisation in 40 daphnids exposed to 100 mg/L loading rate WAF for a period of 48 hours.- It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.PHYSICO-CHEMICAL MEASUREMENTS- The results of the physico-chemical measurements are given in Appendix 2 (attached).- Temperature was maintained at 21.0 °C throughout the test.- There were no treatment related differences for oxygen concentration or pH.VORTEX DEPTH MEASUREMENTS- The vortex depth was recorded at the start and end of the mixing period and was observed to be a lsight dimple at the water surface on each occasion (see Table 3, attached).OBSERVATIONS ON TEST MATERIAL SOLUBILITY- At the start of the mixing period the 100 mg/L loading rate was observed to be a clear colourless water column with globules of test material on the water surface and on the bottom of the mixing vessel.- After 24 hours stirring and after the one hour standing period the 100 mg/L loading rate was observed to remain a clear, colourless solution.- Microscopic inspection of the WAF showed no microdispersions or undissolved test material to be present, therefore a glass wool plug was not used to filter the WAF.

Validity criteria fulfilled:

not specified

Conclusions:

The 48 -hour EL50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for the Testing of Chemicals (1984) No 202, "Daphnia sp, Acute Immobilisation Test and Reproduction Test" referenced as Method C.2 of Commission Directive 92/69/EEC (9which constitutes Annex V of Council Directive 67/548/EEC).

METHODS

Following a preliminary range-finding test, forty daphnids (4 replicates of 10 animals) were exposed to a Water Accommodated Fraction (WAF) of the material, at a single nominal loading rate of 100 mg/L for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

RESULTS

The 48 -hour EL50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Description of key information

The acute toxicity of the test item to Daphnia magna gave a 48 hour EL 50 value of 41 mg/L loading rate WAF with 95% confidence limits of 36 - 47 mg/L loading rate WAF. The No Observed Effect Loading rate at 48 hours was 18 mg/L loading rate WAF. The Lowest Effect Loading rate was considered to be 32 mg/L loading rate WAF (OECD 202 and EU Method C.2).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

41 mg/L

Additional information

Supporting study

Test Guidance

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for the Testing of Chemicals (1984) No 202, "Daphnia sp, Acute Immobilisation Test and Reproduction Test" referenced as Method C.2 of Commission Directive 92/69/EEC (9which constitutes Annex V of Council Directive 67/548/EEC).

Method

Following a preliminary range-finding test, forty daphnids (4 replicates of 10 animals) were exposed to a Water Accommodated Fraction (WAF) of the material, at a single nominal loading rate of 100 mg/L for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

The 48 -hour EL50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Key study

Test Guidance

OECD Guideline No 202 and EC Method C.2

Method

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21 - 22°C under static test conditions. The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

The acute toxicity of the test item to Daphnia magna gave a 48 hour EL 50 value of 41 mg/L loading rate WAF with 95% confidence limits of 36 - 47 mg/L loading rate WAF. The No Observed Effect Loading rate at 48 hours was 18 mg/L loading rate WAF. The Lowest Effect Loading rate was considered to be 32 mg/L loading rate WAF.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, i.e. 0.34 mg/L, to 1.02 mg/L. Chemical analysis of the test preparations at 48 hours showed measured test concentrations to range from less than LOQ to 0.806 mg/L.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the dissolved test item was close to or below the quantifiable limit of the analytical method , the results were based on nominal loading rates only.