o-chlorobenzenethiol

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:

From February 6th to February 9th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.051, 0.128, 0.32, 0.8, 2.0 and 5.0 mg/L (nominal).
- Sampling method: Analytical measurements were performed from the control and at the applied test concentration level at the beginning and at the end of experiment.
- Sample storage conditions before analysis: No storage was necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a nominal concentration of 5.0 mg/L was prepared with the test item and algal growth medium (OECD Medium). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.
- Controls: A concurrent untreated control was examined in parallel to the test item concentrations. A reference control with potassium dichromate is performed at least twice a year to demonstrate satisfactory test conditions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Cultured under standardised conditions (OECD 201). Stock cultures were small algal colonies that are inoculated onto agar regularly. These were transferred to fresh agar medium at least once every two months and were maintained under standardised conditions according to the test guidelines.The pre-culture was intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture 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.

ACCLIMATION
- Acclimation period: no.
- Culturing media and conditions: same as test.
- Any deformed or abnormal cells observed: no.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 – 23.6 ºC

pH:

7.39 – 8.80

Nominal and measured concentrations:

- Nominal: 0.051, 0.128, 0.32, 0.8, 2.0 and 5.0 mg/L
- Measured: 0.031, 0.092, 0.190, 0.420; 1.005 and 2.502 mg/L. As the analytically measured concentration deviated more than 20 per cent from the nominal in most cases during the test, the biological results are based on the measured geometric mean concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks
- Type: covered by air-permeable stoppers, flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- Material, size, headspace, fill volume: glass, size 250 mL, fill volume 100mL test solution.
- Initial cells density: 1 E04
- Control end cells density: 70.83 E04
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 7

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously iluminated
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8418 lux (equivalent to ~114 µE/m2/s), 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 (with observation intervals if applicable) :
- Determination of cell concentrations: manual cell counting using a microscopic method with a counting chamber.
- 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
- Spacing factor for test concentrations: 2.5
- Justification for using less concentrations than requested by guideline:
- Range finding study: yes, at concentrations of 0 (control), 0.1, 1.0, 10.0 and 100.0 mg/L (nominal).
- Test concentrations: 0.051, 0.128, 0.32, 0.8, 2.0, and 5.0 mg/L (nominal).
- Results used to determine the conditions for the definitive study: Significant inhibition was observed at 1 - 100 mg/L in the preliminary test. Chuffed cells could be observed at the nominal concentration of 1.0 mg/L, while thin cells were noted at the two highest levels of 10.0 and 100.0 mg/L (nominal).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.401 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 0.356 - 0.452 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.182 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.196 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.092 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities were observed in the main test. In the preliminary test, chuffed cells could be observed at the nominal concentration of 1.0 mg/L, while thin cells were noted at the two highest levels of 10.0 and 100.0 mg/L (nominal).
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: As the analytically measured concentration deviated more than 20% from the nominal in most cases during the test, the biological results are based on the measured geometric mean concentrations (see table 3 in 'Any other information on results').
- For the lowest tested concentration, the measured concentration was below the Limit of Quantification (LOQ) at the end of the experiment. In order to calculate mean value, where measured concentration was detected, but not quantified, concentration was taken as the half of the Limit of Quantification (LOQ = 0.02 mg/L) according to OECD 23; paragraph 3.3.
- Effect concentrations exceeding solubility of substance in test medium: no
- The ErC50, EbC50 and EyC50 values of the test item could not be calculated, due to the slight effect observed, and therefore, they were determined directly from the raw data.

Results with reference substance (positive control):

- Results with reference substance valid? yes. The values obtained are within the range of laboratory ring test data (see ISO Guideline No. 8692).
- EC50: The date of the last study (Study Code: 16/281-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 13 – 16 September 2016.
- The 72h ErC 50: 0.89 mg/L, (95 % confidence limits: 0.82 – 0.97 mg/L)
- The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
- The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.67 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 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
- Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a= 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 Bonferroni t-Test.

Table 3: Calculation of exposure concentrations

Nominal concentration	Measured concentrations (mg/L)				Geometric mean
	start	24h	48h	72h/end	mg/L
Control	n.d.	n.d.	n.d.	n.d.	-
0.051 mg/L	0.0514	0.0436	0.0394	0.0100*	0.031
0.128 mg/L	0.114	0.096	0.086	0.076	0.092
0.32 mg/L	0.273	0.234	0.199	0.103	0.190
0.8 mg/L	0.699	0.548	0.364	0.224	0.420
2.0 mg/L	1.82	1.27	0.830	0.531	1.005
5.0 mg/L	4.51	3.14	1.95	1.42	2.502

n.d. – not detected *concentration was below the quantification limit. In order to calculate mean value, concentration was taken as the half of the Limit of Quantification (LOQ = 0.02 mg/L) according to OECD 23; see paragraph 3.3.

Table 4: Influence of o-chlorobenzenethiol on the Growth of Pseudokirchneriella subcapitata.

Parameter (0-72 h)	Growth rate (r) [mg/L]		Yield (y) [mg/L]	Biomass (b) [mg/L]
Calculation based on measured geometric mean concentrations
EC50	0.401		0.182	0.196
95 % conf. limits	0.356 – 0.452		0.163 – 0.204	0.174 – 0.221
NOEC	0.092		0.031	0.031
LOEC	0.190		0.092	0.092

Table 5: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period.

Concentration		Growth rate (m) and % inhibition ofm
Nominal	Measured	0–24 h		0–48 h		0–72 h
[mg/L]	[mg/L]	m	%	m	%	m	%
Control	0.0	0.0553^+	0.0	0.0594	0.0	0.0591	0.0
0.051	0.031	0.0538	2.8	0.0598	-0.8	0.0595	-0.7
0.128	0.092	0.0458	17.2	0.0539*	9.3	0.0554*	6.4
0.32	0.190	0.0401*^+	27.4	0.0521*	12.2	0.0517*	12.6
0.8	0.420	0.0345*	37.6	0.0173*^+	70.9	0.0263*	55.5
2.0	1.005	0.0289*	47.8	0.0144*	75.7	0.0096*	83.7
5.0	2.502	0.0000*	100.0	0.0000*	100.0	0.0000*	100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

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

Table 6: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration		Area under the Growth Curves (A) and Percentage Inhibition of A
Nominal	Measured	0–24 h		0–48 h		0–72 h
[mg/L]	[mg/L]	A	%	A	%	A	%
Control	0.0	34.0	0.0	264.0	0.0	1298.0	0.0
0.051	0.031	32.0	5.9	264.0	0.0	1328.0	-2.3
0.128	0.092	24.0	29.4	196.0*	25.8	988.0*	23.9
0.32	0.190	20.0*	41.2	176.0*	33.3	796.0*	38.7
0.8	0.420	16.0*	52.9	48.0*	81.8	132.0*	89.8
2.0	1.005	12.0*	64.7	36.0*	86.4	60.0*	95.4
5.0	2.502	0.0*	100.0	0.0*	100.0	0.0*	100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Table 7: Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration		Concentration
Nominal	Measured	0–72 h
[mg/L]	[mg/L]	Y	%
Control	0.0	69.8	0.0
0.051	0.031	72.0	-3.1
0.128	0.092	53.7*	23.2
0.32	0.190	40.3*	42.2
0.8	0.420	5.7*	91.9
2.0	1.005	1.0*	98.6
5.0	2.502	0.0*	100.0

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Table 8: Cell Number (x 10⁴cell/mL) determined in the Main Experiment

Concentration		Concentration	Number of cells
Nominal	Nominal		0 h	24 h	48 h	72 h
mg/L	mg/L
Control	0.0		1	4	16	70
			1	3	17	80
			1	3	17	72
			1	4	18	62
			1	5	20	76
			1	4	16	65
		Mean	1.00	3.83	17.33	70.83
		SD	0.0	0.8	1.5	6.7
0.051	0.031		1	3	18	65
			1	4	17	78
			1	4	18	76
		Mean	1.00	3.67	17.67	73.00
		SD	0.0	0.6	0.6	7.0
0.128	0.092		1	3	15	58
			1	3	12	64
			1	3	13	42
		Mean	1.00	3.00	13.33	54.67
		SD	0.0	0.0	1.5	11.4
0.32	0.190		1	2	10	40
			1	3	13	44
			1	3	14	40
		Mean	1.00	2.67	12.33	41.33
		SD	0.0	0.6	2.1	2.3
0.8	0.420		1	2	2	6
			1	2	2	7
			1	3	3	7
		Mean	1.00	2.33	2.33	6.67
		SD	0.0	0.6	0.6	0.6
2.0	1.005		1	2	2	2
			1	2	2	2
			1	2	2	2
		Mean	1.00	2.00	2.00	2.00
		SD	0.0	0.0	0.0	0.0
5.0	2.502		1	1*	1*	1*
			1	1*	1*	1*
			1	1*	1*	1*
		Mean	1.00	1.00	1.00	1.00
		SD	0.0	0.0	0.0	0.0

 *   : Thin cells

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met, see 'Overall remarks'.

Conclusions:

The 72h EC50 of the test item on freshwater algae is 0.401 mg/L, the NOEC is 0.092 mg/L and the LOEC is 0.190 mg/L, based on growth rate (r).

Executive summary:

A freshwater algae growth inhibition test was performed on the test item, according to OECD TG 201, under GLP conditions. Based on a rage-finding study where significant inhibition was observed, the test was performed at concentrations of 0.051, 0.128, 0.32, 0.8, 2.0 and 5.0 mg/L test item (nominal) on Pseudokirchneriella subcapitata, alongside concurrent negative controls. Four replicates were performed per concentration and seven for the control. Growth was monitored daily by measuring the number of cells in each culture. Analytical monitoring was performed using a validated method at the start and at the end of the experiment. As the measured values differed more than 20% from the nominal values, the measured concentration values were used to calculate the results. These correspond to 0.031, 0.092, 0.190, 0.420; 1.005 and 2.502 mg/Ltest item (measured).

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range of 0.190 – 2.502 mg/L (measured), therefore the the NOEC was determined as 0.092 mg/L (measured) and LOEC was determined as 0.190 mg/L (measured), based on growth rate. The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 0.092 – 2.502 mg/L (measured). The NOEC was determined as 0.031 mg/L (measured); the LOEC was determined as 0.092 mg/L (measured). The ErC50, EbC50 and EyC50 values of the test item could not be calculated, due to the slight effect observed, and therefore, they were determined directlyfrom the raw data. The 72h EC50 of the test item was 0.401 mg/L, based on growth rate.

Description of key information

Key study. Method according to OECD 201, GLP study. The 72h EC50 of the test item on freshwater algae is 0.401 mg/L, the NOEC is 0.092 mg/L and the LOEC is 0.190 mg/L, based on growth rate (r).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.401 mg/L

EC10 or NOEC for freshwater algae:

0.092 mg/L

Additional information

Key study. Method according to OECD 201, GLP study. Based on a range-finding study, Pseudokirchneriella subcapitata cultures were exposed to 0 (control),0.051, 0.128, 0.32, 0.8, 2.0 and 5.0 mg/L test item (nominal).Four replicates were performed per concentration and seven for the control. Growth was monitored daily by measuring the number of cells in each culture. Analytical monitoring was performed using a validated method at the start and at the end of the experiment. As the measured values differed more than 20% from the nominal values, biological results are based on the measured geometric mean test item concentrations: 0.031, 0.092, 0.190, 0.420; 1.005 and 2.502 mg/L. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the examined concentration range. The 72h EC50 of the test item on freshwater algae was found to be 0.401 mg/L, the NOEC is 0.092 mg/L and the LOEC 0.190 mg/L, based on growth rate (r).