4,4'-bis(chloromethyl)-1,1'-biphenyl

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:

28 June 2016 to 01 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-3.The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc.

Version / remarks:

(Chemical Substance Control Law) 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Provided by sponsor
- Batch/Lot Number: 150701
- Expiration date of the lot/batch: 14 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25 °C), protected from humidity, under inert gas.

Analytical monitoring:

no

Vehicle:

yes

Remarks:

OECD Medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because the test material was very poorly soluble in water, a test solution was prepared using a saturated solution method. A saturated test material solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (OECD Medium) one day before the start of the experiment. This solution was shaken for about 24 hours at approximately 30 °C and then was equilibrated for about 6 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 % saturated solution. A significantly toxic response was not observed during the preliminary concentration Range-Finding Test carried out, therefore only one test concentration at the solubility level of the test material in the test medium (100 % saturated solution) and one control group were tested in a Limit Test.
- Controls: For the untreated control, algal growth medium was inoculated with algal cells (without test material) and was examined in parallel to the test material concentrations. For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested as the reference control at least twice a year to demonstrate satisfactory test conditions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): OECD Medium
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s): The test material solution was a 100 % saturated solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
- Method of cultivation/ breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The preculture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

not specified

Water media type:

other: Reconstituted algal growth medium (OECD medium)

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not measured

Test temperature:

23.0 – 23.2 °C measured in the flask and between 22.6 and 23.5 °C measured within the climate chamber. Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climate chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber.

pH:

7.02 – 8.45. The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test.

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

Because significantly toxic response was not observed during the preliminary concentration range-finding test, only one test concentration at the solubility level of the test item in the test medium (100 % saturated solution) and one control group were tested in the Limit Test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks.
- Type: The flasks were covered with air-permeable stoppers.
- Material, size, headspace, fill volume: The fill volume was 100 mL of test solution.
- Aeration: Not specified, however flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension and the flasks were covered with air-permeable stoppers.
- Initial cells density: Biomass of approximately 10^4 algal cells per mL test medium.
- Control end cells density: The cell density in the control cultures increased by the factor of 69.5 within three days.
- No. of vessels per concentration (replicates): Six replicates per test concentration.
- No. of vessels per control (replicates): Six replicates per test concentration.
- No. of vessels per vehicle control (replicates): Six replicates per test concentration.

GROWTH MEDIUM
- Standard medium used: Reconstituted algal growth medium (OECD medium).


TEST MEDIUM / WATER PARAMETERS
- See Table 1 for composition of OECD medium.

OTHER TEST CONDITIONS
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: 7905 lux (equivalent to ~107 μE/m2/s) continuous illumination which was ensured with fluorescent lamps (with a spectral range of 400 - 700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test material plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group. Test solutions were prepared by appropriate dilution of the stock solution.
- Test concentrations: Preliminary Range-Finding Test nominal concentrations were 0.1, 1.0, 10.0 and 100.0 test material % saturated solutions.
- Results used to determine the conditions for the definitive study: Yes. Because significantly toxic response was not observed during the preliminary concentration Range-Finding test, only one test concentration at the solubility level of the test material in the test medium (100 % saturated solution) and one control group were tested in a limit test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Details on results:

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
There were no morphological deviations of the algal cell during the study in any of the test groups.

AVERAGE SPECIFIC GROWTH RATES
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution.

AREAS UNDER THE GROWTH CURVES
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution.

YIELD
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly the No Observed Effect Concentration (NOEC) was determined as 100 % saturated solution.

Results with reference substance (positive control):

The 72 h ErC50 was 0.84 mg/L, with 95 % confidence limits of 0.77 – 0.92 mg/L.
The 72 h EbC50 was 0.59 mg/L, with 95 % confidence limits of 0.54 – 0.65 mg/L.
The 72 h EyC50 was 0.55 mg/L, with 95 % confidence limits of 0.50 – 0.60 mg/L.

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The ErC50, EbC50 and EyC50 values of the Test Item were determined from the raw data.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software.

For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Table 2.Cell Number (× 10^4 cell/mL) determined in the Main Experiment

Test group		Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	16	67
		1	4	18	73
		1	4	17	72
		1	4	18	69
		1	4	19	70
		1	4	15	66
	Mean	1.00	4.00	17.17	69.50
	SD	0.0	0.6	1.5	2.7
100% saturated solution (nominal)		1	5	19	70
		1	4	18	65
		1	4	18	67
		1	4	17	63
		1	3	17	70
		1	3	18	67
	Mean	1.00	3.83	17.83	67.00
	SD	0.0	0.8	0.8	2.8

Growth rates (µ) and percentage inhibition of µ during the test period

Test group	Growth rate µ and % inhibition of µ
	0 – 24 h		0 – 48 h		0 – 72 h
	µ	%	µ	%	µ	%
Control	0.0573	0.0	0.0592	0.0	0.0589	0.0
100 % saturated solution (nominal)	0.0553*	3.5	0.0600	-1.4	0.0584	0.9

*at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

 

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Growth rate µ and % inhibition of µ
	0 – 24 h		0 – 48 h		0 – 72 h
	µ	%	µ	%	µ	%
Control	36.0	0.0	266.0	0.0	1282.0	0.0
100 % saturated solution (nominal)	34.0	5.6	270.0	-1.5	1264.0	1.4

 

 Yield (Y) and Percentage Inhibition of Y during the Test Period

Test Group	Yield (Y) and % inhibition of Y (0 – 72 h)
	Y	%
Control	68.5	0.0
100 % saturated solution (nominal)	66.0	3.6

 

VALIDITY

The cell density in the control cultures increased by the factor of 69.5 within three days.

The mean coefficient of variation for section-by-section specific growth rates (days 0- 1; 1-2; 2-3) in the control cultures was 7.59 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.93 %. All validity criteria were met, therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 h EC50 was determined to be > 100 % saturated solution; the NOEC and LOEC values based on the growth rate were determined to be 100 % saturated solution and > 100 % saturated solution (nominal), respectively.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the unicellular green alga Pseudokirchneriella subcapitata in accordance with the standardised guidelines OECD 201, EU Method C.3, OCSPP 850.5400 and JMAFF 2-7-3 under GLP conditions.

A toxic response was not observed during the preliminary concentration rangefinding test, therefore a 72 hour limit test was carried out using only one test concentration at the limit of solubility of the test material in the test medium (100 % saturated solution) and one control group. The test design included six replicates at test concentration and six replicates for the untreated control. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test soaftware. The ErC50, EbC50 and EyC50 values of the test material were determined directly from the raw data. Based on the results of this study, the test material had no toxic effect at saturation. The EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Under the conditions of this study, the 72 h EC50 was determined to be > 100 % saturated solution; the NOEC and LOEC values based on the growth rate were determined to be 100 % saturated solution and > 100 % saturated solution (nominal), respectively.

Description of key information

Under the conditions of this study, the 72 h EC50 was determined to be > 100 % saturated solution; the NOEC and LOEC values based on the growth rate were determined to be 100 % saturated solution and > 100 % saturated solution (nominal), respectively.

Additional information

A study was performed to assess the effect of the test material on the growth of the unicellular green alga Pseudokirchneriella subcapitata in accordance with the standardised guidelines OECD 201, EU Method C.3, OCSPP 850.5400 and JMAFF 2-7-3 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

A toxic response was not observed during the preliminary concentration rangefinding test, therefore a 72 hour limit test was carried out using only one test concentration at the limit of solubility of the test material in the test medium (100 % saturated solution) and one control group. The test design included six replicates at test concentration and six replicates for the untreated control. Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test soaftware. The ErC50, EbC50 and EyC50 values of the test material were determined directly from the raw data. Based on the results of this study, the test material had no toxic effect at saturation. The EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Under the conditions of this study, the 72 h EC50 was determined to be > 100 % saturated solution; the NOEC and LOEC values based on the growth rate were determined to be 100 % saturated solution and > 100 % saturated solution (nominal), respectively.