Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental dates: 15 April 2021 to 13 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Test material form:

solid: particulate/powder

Details on test material:

Appearance: beige powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

In the range-finding study a sample of each loading rate WAF was taken for immediate chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. In the main study samples were taken from the control and the 100 mg/L loading rate WAF test group from the bulk test preparation at 0 and 24 hours (fresh media) and from the pooled replicates (Replicates 1 to 4) at 24 and 48 hours (old media) for immediate quantitative analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L. A nominal amount of test item (200 mg) was added to the surface of 2 L of test water to give the 100 mg/L loading rate. After the 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. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour before removal of the aqueous phase or WAF by mid-depth siphoning (the first 100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnids
- Method of breeding: 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.
- Source: In-house laboratory cultures.
- Feeding during test: Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted water (Elendt M4)

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

21 - 22ºC

pH:

7.5 - 7.8

Dissolved oxygen:

8.4 - 8.8 mg O2/L

Nominal and measured concentrations:

Nominal: 100 mg/L (Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines no significant immobilization was observed.)

Measured: Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.0076 to 0.012 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations had declined to less than the LOQ of the analytical method employed, which was determined to be 0.0073 mg/L.
The dissolved test item may have been one or several components of the test item. 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.

Details on test conditions:

In the main study 150 mL vessel 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 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.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

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.

The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a digital thermometer. The light intensity during the light period was measured using an RS Lux meter. The appearance of the test media was recorded daily.

In the range-finding study Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2, 2 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the 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. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour before removal of the WAFs by mid-depth siphoning. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.
In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °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. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

mobility

Details on results:

A single Daphnia was observed to be trapped at the surface in control Replicate 4 at both 24 and 48 hours. There was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours.

Temperature was maintained at approximately 21 ºC to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 839 to 870 lux.

At the start of each mixing period the 100 mg/L preparations were observed to be a clear colorless media column with many particles of test item visible floating at the surface. After 23 hours stirring and a 1-Hour standing period the 100 mg/L preparations were observed to remain as at the start of the mixing. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 100 mg/L preparations were observed to be clear, colorless solutions.
At the start and throughout the test, all control and test solutions were observed to be clear colorless solutions.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.0076 to 0.012 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations had declined to less than the LOQ of the analytical method employed, which was determined to be 0.0073 mg/L.
The dissolved test item may have been one or several components of the test item. 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.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal range for this reference item (EC50(48 h):0.50 mg/L, NOEC:0.32 mg/L).

Any other information on results incl. tables

The definitive test was considered to be valid given that none more than 10% 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 equal to or greater than 3 mg/L in the control and test vessels.

 

Results range-finding test

No immobilization or sub-lethal effects of exposure were observed throughout the test.
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 significant immobilization was observed.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 0.023 to 0.20 mg/L. Chemical analysis of the test preparations at 48 hours showed measured test concentrations to range from test item not detected to 0.0040 mg/L, indicating that the test item was not stable under test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of an acute daphnia toxicity study performed according to OECD Guideline 202 and GLP principles, the NOEL of BTMR was concluded to be 100 mg/L (loading rate WAF).

Executive summary:

A study was performed with BTMR to assess the acute toxicity of the test item to Daphnia magna, according to OECD Guideline 202 and GLP principles. Due to the low aqueous 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).
Following a preliminary range-finding test and initial experiment, 20 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 approximately 21°C to 22°C under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.0076 to 0.012 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations had declined to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0073 mg/L.
The dissolved test item may have been one or several components of the test item. 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.
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 (NOEL) rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

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