Sulfonic acids, shale-oil, sodium salts

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:

initiated: 2016-08-22, experimental: 2017-01-18 to 2017-03-14 / positive control 2016-10-20 to 2016-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

recommended study/method

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

yes

Remarks:

Temperature was measured from 19 - 22 ºC. This was a deviation to the Study Plan which indicated 18 °C - 22 °C ± 1 °C. This deviation was considered to have not affected the validity of the study given that no adverse effects to exposure were observed.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

yes

Remarks:

Temperature was measured from 19 - 22 ºC. This was a deviation to the Study Plan which indicated 18 °C - 22 °C ± 1 °C. This deviation was considered to have not affected the validity of the study given that no adverse effects to exposure were observed.

GLP compliance:

yes (incl. QA statement)

Remarks:

MHRA, date ofissue: 28/10/2016

Specific details on test material used for the study:

technical grade: aqueous solution (31% dry matter in water)

Analytical monitoring:

yes

Details on sampling:

RANGE-FINDING TEST
In the initial range-finding test, a sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

DEFINITIVE TEST
Samples were taken from the control and the 100 mg active ingredient/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in Envigo Study Report: NF11MJ, Annex 4.

Vehicle:

no

Details on test solutions:

Reconstituted water (Elendt M7) used for both the range-finding and definitive test:
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.

Test type:

not specified

Water media type:

other: reconstituted water (Elendt M7)

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure.

Hardness:

NA

Test temperature:

19 - 22 °C

pH:

7.9 ± 0.3

Dissolved oxygen:

8.1 - 9.1 mg/L

Salinity:

NA

Conductivity:

NA

Nominal and measured concentrations:

- range-finding test: 0.10, 1.0, 10 and 100 mg/L
- definite test, nominal concentration: 100 mg active ingredient/L

Details on test conditions:

Based on the results of the range-finding tests, a "limit test" was conducted at a concentration of 100 mg active ingredient/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed. A nominal amount of test item (323 mg) was dissolved in test water and the volume adjusted to 1000 mL to give the 100 mg active ingredient/L test concentration. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparation was verified by chemical analysis at 0 and 48 hours

EXPOSURE CONDITIONS
In the definitive test, 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test, five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 °C to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The 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. The test preparations were not renewed during the exposure period.

TEST ORGANISM OBSERVATIONS
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

WATER QUALITY CRITERIA
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

VERIFICATION OF TEST CONCENTRATIONS
Samples were taken from the control and the 100 mg active ingredient/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

STATISTICAL ANALYSIS
An estimate of the EC50 values was given by inspection of the immobilization data.

VALIDATION CRITERIA
The results of the test are considered valid if the following performance criteria are met:
- No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
- The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

MAJOR COMPUTERIZED SYSTEMS
Delta control system, Building management

Reference substance (positive control):

yes

Remarks:

potassium dichromate (Envigo Study Number XT01HS)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TESTS
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding tests are given in Table 1 and 2. No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L in the initial range-finding test, or 10 and 100 mg active ingredient/L in the second range-finding test. Based on this information, a single test concentration of four replicates, of 100 mg active ingredient/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed. Chemical analysis of the 100 mg/L test preparation, corresponding to 31 mg active ingredient/L, at 0 hours (see Annex 4) showed a measured test concentration of 98% of nominal concentration. There was no significant change in the measured concentration at 48 hours indicating that the test item was stable under test conditions.

DEFINITIVE TEST
Verification of Test Concentrations: Analysis of the test preparations at 0 and 48 hours (see Annex 4) showed measured test concentrations to range from 91% to 94% of nominal concentration, and so the results are based on the nominal test concentration.

Immobilization Data: Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 3. There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg active ingredient/L for a period of 48 hours. Inspection of the immobilization data gave the following results:
24 h EC50 > 100 mg/L
48 h EC50 > 100 mg/L
24 h NOEC = 100 mg/L
48 h NOEC = 100 mg/L.

Sub-Lethal Effects: No sub-lethal effects of exposure were observed throughout the test.

Validation Criteria: The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress, and that the oxygen concentration at the end of the test was greater than 3 mg/L in the control and test vessels.

Water Quality Criteria: The results of the water quality measurements are given in Table 4. Temperature was maintained at 19 °C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on Test Item Solubility: At the start and throughout the test, the control was observed to be a clear, colorless solution; the test preparations were observed to be clear, brown solutions.

Results with reference substance (positive control):

A positive control (Envigo Study Number XT01HS) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Exposure conditions for the positive control were similar to those in the definitive test. Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
24 h EC50 = 1.3 mg/L (95% CL: 1.0 - 1.8)
48 h EC50 = 1.2 mg/L (95% CL: 1.1 - 1.3
24 h NOEC = 1.0 mg/L
48 h NOEC = 0.56 mg/L
24 h LOEC = 1.8 mg/L
48 h LOEC = 1.0 mg/L

The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

Immobilization data, 24 h: Binomial Distribution method
Immobilization data, 48 h: Trimmed Spearman-Karber method

All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg active ingredient/L. The No Observed Effect Concentration was 100 mg active ingredient/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed 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

Following preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100 mg active ingredient/L for 48 hours at a temperature of 19 °C to 22 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 91% to 94% of the nominal concentration, and so the results are based on the nominal test concentration. Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg active ingredient/L. The No Observed Effect Concentration was 100 mg active ingredient/L.

Description of key information

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed 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

Following preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100 mg active ingredient/L for 48 hours at a temperature of 19 °C to 22 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 91% to 94% of the nominal concentration, and so the results are based on the nominal test concentration. Exposure of Daphnia magna to the test item gave EC50 values of greater than 100 mg active ingredient/L. The No Observed Effect Concentration was 100 mg active ingredient/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The test item contains 31% active ingredient. The active ingredient concentration in the test samples was determined by high performance liquid chromatography with tandem quadrapole mass spectrometery (HPLC-MS/MS) using an external standard. The active ingredient gave a chromatographic profile consisting of several peaks. The method was developed and implemented by the Department of Analytical Services, Envigo Research Limited, Shardlow, UK.