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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

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.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) 761/2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

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:

Main study:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. A further sample of each test group containing no algal cells was incubated alongside the test from which samples were taken at 24 and 48 hours for immediate analysis.
Range-finder study:
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were analyzed on the day of sampling.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
- Culturing media and conditions: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. The culture medium used for the validation of mixing period trial, initial experiment, range-finding test and definitive test was the same as that used to maintain the stock culture.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Purified deionized water

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

7.4 - 8.9

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured: Chemical analysis of the 1.0, 3.2, 10, 32 and 100 mg/L test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of less than the LOQ of the analytical method employed, determined to be 0.0070 mg/L, were obtained.
This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.
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:

Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Nominal amounts of test item (10, 32, 20, 64 and 200 mg) were each separately added to the surface of 10, 10, 2, 2 and 2 L of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium 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 95 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the 100 mg/L loading rate WAF indicated that dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (first 100 mL discarded). Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions of test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.1 mL) to give an initial nominal cell density of 5.00 x 10E3 cells/mL.
Glass conical flasks of 250 mL were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.20 x 10E6 cells per mL. Inoculation of 500 mL of test medium with 2.1 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10E3 cells per mL and had no significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated at 24±1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically, and any abnormalities recorded.

The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure. The appearance of the test media was recorded daily. The vortex depth was recorded at the start and end of the mixing period.

Range-finding test
The range-finding test was conducted by exposing Raphidocelis subcapitata cells to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 72 hours.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 L of culture medium to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium 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 96 hours and the mixtures allowed to stand for 1 hour. The stirring period was 1 hour longer than instructed due to technician error, however as the stirring period was at least as long as required and the preparation was comparable to that produced in the preliminary validation of mixing period, the preparation was considered to be acceptable for use. Microscopic observations made on the WAFs indicated that dispersed test item was present in the water column of the 100 mg/L preparation and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (first 100 mL discarded). Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions of test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (1.7 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined. The flasks were then plugged with polyurethane foam bungs and incubated at 24±1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined.

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Detailed results are attached below. The growth rate and yield of Raphidocelis subcapitata were not affected by the presence of BTMR over the 72-Hour exposure period.

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

The pH value of the control cultures was observed to increase from pH 7.4 at 0 hours to pH 8.4 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. Temperature was maintained at 24±1ºC throughout the test. The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

At the start of the mixing period the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF preparations were observed to be clear colorless media columns with test item at the media surface. After 95 hours stirring, all loading rate WAFs were observed to have formed clear, colorless media columns with test item at the surface and suspended within the media column. Following a 1 hour standing period, the 1.0, 3.2, 32 and 100 mg/L loading rates had formed clear colorless media columns with test item at the surface whilst the 10 mg/L loading rate had formed a clear colorless media column with test item at the media surface and particles settled on the bottom of the mixing vessel. Micro-dispersions of test item were observed to be present in the 100 mg/L loading rate WAF and as such all loading rates were filtered through a glass wool plug (first 100 mL discarded). Microscopic observations after filtration showed there to be no micro-dispersions of test item present.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test cultures were observed to be green dispersions.

Chemical analysis of the 1.0, 3.2, 10, 32 and 100 mg/L test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of less than the LOQ of the analytical method employed, determined to be 0.0070 mg/L, were obtained.
This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ. 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):

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.5 mg/L
EyC50 (0 to 72 hour) : 0.44 mg/L; 95% confidence limits 0.37 to 0.52 mg/L
No Observed Effect Concentration based on growth rate: 0.125 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.25 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and test group using the Dunnett’s Multiple t-test Procedure. There were no statistically significant differences (P=0.05), between the control and the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test groups and therefore the NOEL based on growth rate was 100 mg/L loading rate WAF.
Statistical analysis of the yield data was carried out. There were no statistically significant differences between the control and the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF (P=0.05 and, therefore the NOEL based on yield was 100 mg/L loading rate WAF.

Any other information on results incl. tables

Preliminary investigational work indicated that there was a significant increase in the amount of dissolved test item when the preparation period was extended to 96 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 95 hours followed by a 1-Hour settlement period.

Range-finding test
The results showed no effect on growth at 1.0, 10 and 100 mg/L loading rate WAF however, yield was observed to have been reduced. Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test. Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 0.0033 to 0.018 mg/L and 0.00097 to 0.0023 mg/L at 72 hours indicating that the test item was unstable under test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of BTMR on the growth of Raphidocelis subcapitata has been investigated according to  OECD Guideline 201 and GLP principles and resulted in a NOEL of 100 mg/L (loading rate WAF).

Executive summary:

A study was performed to assess the effect of BTMR on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) according to  OECD Guideline 201 and GLP principles.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item. Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24±1°C. Samples of the algal populations were removed daily and cell concentrations were determined for each control and treatment group.

Chemical analysis of the 1.0, 3.2, 10, 32 and 100 mg/L test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0070 mg/L, were obtained.
This analytical result does not indicate absence of test item in the solution, but any dissolved test item present was at a concentration below the LOQ. Measured test concentrations below the limit of determination of the analytical method were determined to be 0.0010 mg/L to 0.0065 mg/L were obtained at 0 hours.
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 Raphidocelis subcapitata to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate (NOEL) was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.