Reaction mass of Benzeneethanol, 2-chloro-α-(1-chlorocyclopropyl)-α-(chloromethyl)- and Oxirane, 2-(1-chlorcyclopropyl)-2-[(2-chlorphenyl)methyl]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-UV/VIS

Details on sampling:

Sampling schedule:
- Control: at 72 hours
- Test concentration/s : 0.1, 0.32, 1.0, 3.2 and 10 mg/L at 0, 24, 48 and 72 hours
Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations
A stock solution was prepared to give the desired series of test concentrations. 33.0 mg of the test item (= 10 mg/L active ingredient) were added to 2 litres of dilution water, treated for 60 seconds at 8000 rpm with an ultra turrax and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7-12 µm. The pH was measured to be pH 7.9.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.794 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.

Controls: In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
- Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977)) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.
- Age of inoculum (at test initiation): 3 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH, corresponding to 22.5 mg/L CaCO3. (final nutrient medium )

Test temperature:

21 °C to 24 °C

pH:

7.9 - 9.0

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

Test concentrations: 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2 and 10 mg/L (active ingredient)
Effective concentrations of the active ingredient ranged from 59.7 % to 67.4 % of nominal values at 0 hours, from 50.9 % to 57.8 % of nominal values at 24 hours, from 42.5 % to 48.4 % of nominal values at 48 hours and from 26.8 % to 39.9 % of nominal values at 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
- culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
- Light intensity: A light intensity ranging from 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.
- Cell density measurements: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
- Experimental design: 8 test concentrations plus 1 control, 3 replicates per concentration / control, additionally highest test concentration without algae
- Initial cell density in the test cultures approximately 5000 cells per millilitre
- concentration/s: 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2 and 10 mg/L (active ingredient)
- Method of administration: stock solution
- Duration of exposure: 72 hours
- Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.57 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The results are expressed in terms of geometric mean measured concentrations of the active ingredient. Effective concentrations of the active ingredient ranged from 59.7 % to 67.4 % of nominal values at 0 hours, from 50.9 % to 57.8 % of nominal values at 24 hours, from 42.5 % to 48.4 % of nominal values at 48 hours and from 26.8 % to 39.9 % of nominal values at 72 hours.
NOEC/LOEC (72 h) determination by statistical methods for growth rate does not appear to be meaningful. For growth rate (72 h) the statistical NOEC and LOEC values are 0.0450 mg/L and 0.1534 mg/L, respectively. In this case an effect of < 4 % inhibition was determined as LOEC value, whereas from the biological point of view the result of only values >10% inhibition show biological effects. In addition, the values indicating significance to the control, fluctuate. The next higher concentration 0.4529 mg/L shows no significance to the control, while the following higher concentration 1.6092 mg/L indicates significance to the control. The corresponding inhibition values are 5.8 % for 0.4529 mg/L and 44.3 % for 1.6092 mg/L. These values correspond much better to the effect values and to the determined EC10 and EC50-values. For these reasons the NOEC/LOEC values were determined as 0.4529 mg/L (= 0.45 mg/L) for the NOEC and 1.6092 mg/L (= 1.6 mg/L) for the LOEC.

Reported statistics and error estimates:

All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.

NOEC and LOEC were determined according to Williams test and Welch-t test.

Validity criteria fulfilled:

yes

Remarks:

factor of biomass parameter >16; The mean of the replicate coefficients of variation in the section-by-section growth <35 %; coefficient of variation of the mean specific growth rate replicates in the control <7 %

Conclusions:

An analysis of the growth rate of the algal population within the 72 h exposure period gave the following results regarding the toxicity of the test substance towards algae: EC10 = 0.57 mg/L; EC50 = 1.8 mg/L; NOEC= 0.45 mg/L and LOEC = 1.6 mg/L.
The results are expressed in terms of geometric mean measured concentrations of the active ingredient. Effective concentrations of the active ingredient ranged from 59.7 % to 67.4 % of nominal values at 0 hours, from 50.9 % to 57.8 % of nominal values at 24 hours, from 42.5 % to 48.4 % of nominal values at 48 hours and from 26.8 % to 39.9 % of nominal values at 72 hours.

Executive summary:

A study was performed to assess the adverse effects on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range finding test: 0.0001, 0.001, 0.01, 0.1, 1.0 and 10 mg/L.

Exponentially growing algal cells were exposed for a period of 72 h to a range of concentrations, nominally 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2 and 10 mg/L of the active ingredient of the test substance. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and a folded filter. The cell densitites were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth rate relative to control cultures grown under identical conditions. The 72 h EC10 and EC50 were calculated by probit analysis. During the test a temperature range of 21 - 24 °C was maintained in the test vessels.The pH was measured at the beginning of the test and after 72 hours of exposure. The maintenance of the test item concentrations was proved by analytical measurements.In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test. Chemical analysis and pH measurement at the end of the test were performed using a replicate of the test concentrations and the control vessels. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations. An EC10 of 0.57 mg/L, an EC50 of 1.8 mg/L, a NOEC of 0.45 mg/L and a LOEC of 1.6 mg/Lwere determined.

Description of key information

A study was performed to assess the adverse effects on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range finding test: 0.0001, 0.001, 0.01, 0.1, 1.0 and 10 mg/L.

Exponentially growing algal cells were exposed for a period of 72 h to arange of concentrations, nominally 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2 and 10 mg/L of the active ingredient of the test substance.Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and a folded filter.The cell densitites were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth rate relative to control cultures grown under identical conditions. The 72 h EC10 and EC50 were calculated by probit analysis.During the test a temperature range of 21 - 24 °C was maintained in the test vessels.The pH was measured at the beginning of the test and after 72 hours of exposure.The maintenance of the test item concentrations was proved by analytical measurements.In order to avoid an impairment of the test system, an additional replicate was used for analysis and pH measurement at the beginning of the test. Chemical analysis and pH measurement at the end of the test were performed using a replicate of the test concentrations and the control vessels. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, a test flask at the highest test concentration without algae was run in parallel to the geometric series of test concentrations. An EC10 of 0.57 mg/L, an EC50 of 1.8 mg/L,aNOEC of 0.45 mg/L and a LOEC of 1.6 mg/Lwere determined.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.8 mg/L

EC10 or NOEC for freshwater algae:

0.57 mg/L