Reaction Products of Dimethyl hydrogen phosphite and Alcohols C14-18, Even, Linear

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 February 2015 to 05 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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 TESTS
- A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the tability of the test item under test conditions.
- All samples were stored frozen prior to analysis.
- Only concentrations within the range to be used for the definitive test were analysed.

DEFINITIVE TEST
- Samples were taken from the control and each loading rate WAF test goup at 0 hours (taken from bulk test preparation) and 48 hours (replicates R1 to R4 pooled) for quantitative analysis.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

TEST WATER
- Reconstituted water (ISO medium) used for both the range-finding and definitive test is defined in Appendix 4 (attached).

PROCEDURE
- Due to the low solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

VALIDATION OF MIXING PERIOD
- Preliminary work (see Appendix 5, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

RANGE-FINDING TEST
- Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
- Prior to use, the test item was melted at 50 °C for 2 hours.
- Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively.
- After addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour.
- Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove 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 1.0, 10 and 100 mg/L loading rate WAFs.
- Microscopic observations were performed on the WAFs after filtering and showed no undissolved test item remained in the water column.

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 mortalities or sub-lethal effects of exposure were observed.
- Prior to use, the test item was melted at 50 °C for 2 hours.
- Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively.
- After addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour.
- Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove 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 1.0, 10 and 100 mg/L loading rate WAFs.
- Microscopic observations were performed on the WAFs after filtering and showed no undissolved test item remained in the water column.

Test organisms (species):

Daphnia magna

Details on test organisms:

- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 2, attached) in a temperature controlled room at approximately 21°C.
- The lighting cycle was controlled to give 16 hours light and 8 hours darkness with 20 minute dawn and dusk periods.
- Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Culture conditions ensured that reproduction was by parthenogenesis.
- Gravid adults were isolated the day before initiation of the test so that 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 were 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:

None

Hardness:

250 mg/L total theoretical hardness as CaCO3

Test temperature:

21 °C

pH:

7.6 to 8.8

Dissolved oxygen:

7.5 to 8.8 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

RANGE-FINDING TEST
- Nominal loading rates of 1.0, 10 and 100 mg/L

DEFINITIVE TEST
- Nominal loading rate of 100 mg/L

Details on test conditions:

RANGE-FINDING TESTS
- Daphnids (10) were placed in each test and control vessel and maintained at a temperature controlled room at approximately 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.

DEFINITIVE TEST
- 250 mL glass jars containing approximately 200 mL of test preparation were used.
- At the start of the test daphnids (5) were placed in each test and control vessel at random.
- Four replicate test and control vessels were prepared.
- Test vessels were covered to reduce evaporation and maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light (342 to 407 lux) 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.
- Test preparations were not renewed during the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate conducted between 23 February 2015 and 25 February 2015 (see Appendix 3, attached)

Duration:

24 h

Dose descriptor:

NOELR

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 item during the range-finding test are given in Table 1 (attached).
- No immobilisation was observed at 1.0, 10 and 100 mg/L loading rate WAF.
- Based on this information, a single loading rate of four replicates of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no immobilisation or adverse reactions to exposure was observed.
- Chemical analysis of the 100 mg/L loading rate at 0 and 48 hours (see Appendix 6, attached) showed measured concentrations of 15.3 and 22.3 mg/L respectively and indicated that the test item was stable under the stated experimental conditions.

DEFINITIVE TEST
- Analysis of test preparations at 0 and 48 hours (see Appendix 6, attached) showed measured concentrations of 8.2 and 6.8 mg/L respectively.
- Dissolved test item may ahve been one or several components of the test item and, given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.
- Lower measured concentrations were observed in the definitive test when compared to the range-finding test. This was considered to be due to the efficiency of the filtration method. However, the lower values were not considered to have had an impact on the outcome of the test.

IMMOBILISATION DATA
- Cumulative immobilisation data from exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- There was no immobilisation in 20 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.
- A single immobilised daphnid was observed in the control group at 48 hours. However, no more than 10 % immobilisation occurred and no impact on the outcome of the test was considered to have taken place.

Results with reference substance (positive control):

- Results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

- An estimate of the EL50 values was given by inspection of the immobilisation data.

VALIDATION CRITERIA

- No more than 10 % of the control daphnids showed immobilisation or other signs of disease or stress.

- Oxygen concentration at the end of the test was ≥ 3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA

- Results of the water quality measurements are given in Table 3 (attached).

- Temperature was maintained at 21 °C throughout the test.

- There were no treatment-related differences for oxygen concentration or pH.

VORTEX DEPTH MEASUREMENTS

- Vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

OBSERVATIONS ON TEST ITEM SOLUBILITY

- Observations on the test media were carried out during mixing and testing of the WAF.

- At the start of the mixing period the 100 mg/L loading rate was observed to be a clear, colourless, water column with lumps of test item on the surface.

- After 23 hours stirring, the 100 mg/L loading rate was observed to be a clear, colourless, water column with flakes and lumps of test item throughout and on the surface.

- After a 1 -hour standing period the 100 mg/L loading rate was observed to be a clear, colourless, water column with flakes of test item suspended throughout.

- Examination of the WAF showed that undissolved test item was suspended in the water column and it was therefore considered justifiable to remove the WAF by filtering through a glass wool plug (2 -4 cm in length).

- Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed test item.

- During the test the 100 mg/L loading rate was observed to be a clear, colourless, solution.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect loading rate was 100 mg/L loading rate WAF.

Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

METHODS

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

RESULTS

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours showed measured concentrations of 8.2 and 6.8 mg/L respectively.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

CONCLUSION

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect loading rate was 100 mg/L loading rate WAF.

Description of key information

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect loading rate was 100 mg/L loading rate WAF (OECD 202 and EU Method C.2).

Additional information

GUIDELINE

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

METHODS

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

RESULTS

Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours showed measured concentrations of 8.2 and 6.8 mg/L respectively.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

CONCLUSION

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect loading rate was 100 mg/L loading rate WAF.