Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

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:

The range-finding test was conducted between 28 May 2002 and 31 May 2002 and the definitive test between 5 June 2002 and 8 June 2002.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test materials has shown that a mixing period of 24 - 48 hours is sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

The concentration, homogeneity and stability of the test material in the test preparations were not determined at the request of the Sponsor.

Vehicle:

no

Details on test solutions:

Range-finding tests
The loading rates to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Scenedesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.

The test was conducted in 250 ml glass conical flasks plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was prepared as a WAF.

Amounts of test material (100 and 200 mg) were each separately dispensed onto the surface of 10 and 2 litres of culture medium via a plastic disposable syringe to give the 10 and 100 mg/l loading rates respectively. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped after 24 hours and the mixtures allowed to stand for 1 hour. A small amount of each of the WAFs was then removed and checked microscopically for the presence of micro-dispersions or globules of test material. No test material was observed to be present and it was therefore considered appropriate to remove the WAF or aqueous phase by mid-depth siphoning. A wide bore glass tube, covered at one end with Parafilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Parafilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. An aliquot (500 ml) of each of the WAFs was separately inoculated with algal suspension (2.5 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.

Given that significant inhibition of growth was observed at all test concentrations a second range-finding test was conducted at concentrations of 0.10, 1.0 and 10 mg/l loading rate WAF.

Amounts of test material (2.0, 20 and 100 mg) were each separately weighed onto glass slides and washed onto the surface of 20, 20 and 10 litres of culture medium to give the 0.10, 1.0 and 10 mg/l loading rates respectively. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped after 24 hours and the mixtures allowed to stand for 1 hour. A small amount of each of the WAFs was then removed and checked microscopically for the presence of micro-dispersions or globules of test material. No test material was observed to be present and it was therefore considered appropriate to remove the WAF or aqueous phase by mid-depth siphoning. A wide bore glass tube, covered at one end with Parafilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Parafilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 0.10, 1.0 and 10 mg/l loading rate WAFs. An aliquot (500 ml) of each of the WAFs was separately inoculated with 2.5 ml algal suspension to give the required test concentrations of 0.10, 1.0 and 10 mg/l loading rate WAF.
The control groups were maintained under identical conditions but not exposed to the test material.

At the start of the range-finding tests a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer II Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron- Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer II Particle Counter.

Definitive test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.313, 0.625, 1.25, 2.5, 5.0 and 10 mg/l loading rate WAF.

Experimental Preparation
Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
Amounts of test material (3.13, 6.25, 12.5, 25, 50 and 100 mg) were each separately weighed onto glass slides and washed onto the diluent surface of 10 litres of culture medium to give the 0.313, 0.625, 1.25, 2.5, 5.0 and 10 mg/l loading rates respectively. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. These were stirred for 24 hours. The stirring was stopped after 24 hours and the mixtures allowed to stand for 1 hour. A small amount of each of the WAFs was removed and checked microscopically for the presence of micro-dispersions or globules of test material. No test material was observed to be present and it was therefore considered appropriate to remove the WAF or aqueous phase by mid-depth siphoning. A wide bore glass tube, covered at one end with Parafilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Parafilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 0.313, 0.625, 1.25, 2.5, 5.0 and 10 mg/l loading rate WAFs. An aliquot (1 litre) of each of the WAFs was separately inoculated with algal suspension (5.0 ml) to give the required test concentrations of 0.313, 0.625, 1.25, 2.5, 5.0 and 10 mg/l loading rate WAF.
The use of glass slides enabled small amounts of test material to be weighed out accurately.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Scenedesmus subspicatus is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism in freshwater ecosystems.

Test Species
The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria. Cultures were maintained in the laboratory by the periodic replenishment of culture medium . The culture was maintained in the laboratory at a temperature of 21 ± 1ºC under continuous illumination (intensity approximately 7000 lux) and constant aeration.


- Culturing media and conditions:
Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined in Appendix 1.

Appendix 1:
Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. The prepared media was sterilised by 0.2 µm membrane filtration and stored in darkness.

- Any deformed or abnormal cells observed:
Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Observations on test material solubility
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period the control, 0.313, 0.625, 1.25 and 2.5 mg/l loading rate WAF test cultures were observed to be bright green dispersions. The 5.0 mg/l loading rate WAF test cultures were observed to be pale green dispersions whilst the 10 mg/l loading rate WAF test cultures were observed to be very pale green dispersions.

Hardness:

Not recorded.

Test temperature:

The temperature within the incubator was recorded daily. Temperature was maintained at 24 ± 1ºC throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The pH values of each test and control flask are in Table 3 under results incl. tables section .
The pH values of the control cultures (see Table 3 under results incl. tables section) were observed to increase from pH 7.4 at 0 hours to pH 7.9 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.
The test material vessels showed an increase in pH over the 72-Hour test period following a concentration dependent pattern with the lower test material concentrations exhibiting a greater increase in pH (see Table 3). This effect was considered to be due to there being greater numbers of viable cells in the lower test concentrations and hence greater utilisation of carbonates and bicarbonates from photosynthesis/respiration

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The results showed no effect on growth at the 0.10 mg/l loading rate WAF. However, growth was observed to be reduced at the 1.0, 10 and 100 mg/l loading rate WAFs. Based on this information and given the flat response observed during the second range-finding test loading rates of 0.313, 0.625, 1.25, 2.5, 5.0, and 10 mg/l, using a stirring period of 24 hours followed by a 1-Hour standing period, were selected for the definitive test.

Details on test conditions:

Exposure conditions
As in the range-finding test 250 ml glass conical flasks were used. Three flasks each containing 100 ml of test preparation were used for the control and each treatment group. The control group was maintained under identical conditions but not exposed to the test material. Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.45 x 106 cells per ml. Inoculation of 1 litre of test medium with 5.0 ml of this algal suspension gave an initial cell density of 104 cells per ml and had no significant dilution effect. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron(R) Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm for 72 hours. Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer II Particle Counter.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

other: EbL50

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL not stated

Duration:

72 h

Dose descriptor:

other: ErL50

Effect conc.:

63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.313 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not specified

Details on results:

Range-finding Tests
The cell counts and percentage inhibition of growth values from the exposure of Scenedesmus subspicatus to the test material during the range-finding tests are given in Table 1 and 2 under results incl. tables section. The results showed no effect on growth at the 0.10 mg/l loading rate WAF. However, growth was observed to be reduced at the 1.0, 10 and 100 mg/l loading rate WAFs. Based on this information and given the flat response observed during the second range-finding test loading rates of 0.313, 0.625, 1.25, 2.5, 5.0, and 10 mg/l, using a stirring period of 24 hours followed by a 1-Hour standing period, were selected for the definitive test.

Definitive Test
Growth data
From the data given in Tables 3 and 4, it is clear that both the growth (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period. The mean cell densities versus time for the definitive test are presented in Figure 1. The percentage inhibition values (IA), (Table 4) were plotted against loading rate (Figure 2), a line fitted by eye and the EL*50 value with respect to the area under the growth curve, EbL50 (72h) read from the graph. Percentage reductions in growth rate and the EL*50 value with respect to growth rate, ErL50 value (0 - 72 hours) are calculated and the results given in Table 4 and Figure 2.

Accordingly the following results were determined from the data:
EbL*50 (72 h) : 3.5 mg/l loading rate WAF
ErL*50 (0 - 72 h) : 63 mg/l loading rate WAF**
where EbL50 is the loading rate that reduced biomass by 50% and ErL50 is the loading rate that reduced specific growth rate by 50%.
It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.
* EL = Effective Loading rate
** Value obtained by extrapolation of the growth curve data as no concentration tested resulted in greater than 50% inhibition of growth
Statistical analysis of the area under the growth curve data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control and 0.313 mg/l loading rate WAF test group (P greater than or equal to 0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading rate" (NOEL) was 0.313 mg/l loading rate WAF.

The following data show that the cell concentration of the control cultures increased by a factor of 69 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours:
Mean cell density of control at 0 hours : 1.28 E+04 cells per ml
Mean cell density of control at 72 hours : 8.89 E+05 cells per ml

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a slight dimple at the media surface (see Table 5).

Results with reference substance (positive control):

not stated

Reported statistics and error estimates:

Statistical analysis
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the area under the growth curve data at 72 hours for the control and all the loading rate WAF test groups to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1996).

Table 1: Cell Densities and Percentage Inhibition of Growth from the Initial Range-finding Test

Nominal Loading Rate (mg/l)		Cell Densities*(cells per ml)
		0 Hours	72 Hours	% Inhibition (area under curve at   72 h)
Control	R1	1.40E+04	4.10E+05
	R2	9.06E+03	4.45E+05	-
	Mean	1.15E+04	4.28E+05
10	R1	1.27E+04	7.13E+04
	R2	8.70E+03	7.13E+04	85
	Mean	1.07E+04	7.13E+04
100	R1	1.13E+04	3.97E+03
	R2	9.85E+03	3.28E+03	102
	Mean	1.06E+04	3.62E+03

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁– R₂= Replicates 1 and 2

 

Table 2: Cell Densities and Percentage Inhibition of Growth from the Second Range-finding Test

Nominal Loading Rate (mg/l)			Cell Densities*(cells per ml)
			0 Hours		72 Hours		% Inhibition (area under curve at   72 h)
Control		R1		8.78E+03		7.25E+05
		R2		8.46E+03		7.15E+05		-
		Mean		8.62E+03		7.20E+05
0.10		R1		9.91E+03		6.46E+05
		R2		8.65E+03		6.35E+05		11
		Mean		9.28E+03		6.41E+05
1.0		R1		9.74E+03		4.78E+05
		R2		9.25E+03		4.58E+05		36
		Mean		9.50E+03		4.68E+05
10		R1		9.12E+03		1.95E+05
		R2		8.50E+03		1.91E+05		74
		Mean		8.81E+03		1.93E+05

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.R₁– R₂= Replicates 1 and 2

Table 3: Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.4	1.29E+04	6.59E+04	1.39E+05	8.91E+05	7.9
	R2	7.4	1.29E+04	6.52E+04	2.15E+05	8.93E+05	7.9
	R3	7.4	1.26E+04	5.81E+04	1.72E+05	8.83E+05	7.9
	Mean		1.28E+04	6.30E+04	1.75E+05	8.89E+05
0.313	R1	7.4	1.29E+04	5.16E+04	2.42E+05	8.74E+05	8.4
	R2	7.4	1.25E+04	5.20E+04	2.07E+05	8.85E+05	8.0
	R3	7.4	1.24E+04	5.84E+04	1.63E+05	8.68E+05	8.1
	Mean		1.26E+04	5.40E+04	2.04E+05	8.75E+05
0.625	R1	7.4	1.30E+04	5.70E+04	2.31E+05	6.55E+05	8.3
	R2	7.4	1.20E+04	5.44E+04	1.79E+05	6.49E+05	8.2
	R3	7.4	1.27E+04	5.46E+04	1.92E+05	6.33E+05	8.2
	Mean		1.26E+04	5.53E+04	2.01E+05	6.45E+05
1.25	R1	7.4	1.54E+04	4.97E+04	1.71E+05	6.14E+05	8.0
	R2	7.4	1.58E+04	4.46E+04	1.64E+05	6.23E+05	8.0
	R3	7.4	1.37E+04	4.63E+04	1.94E+05	6.14E+05	8.0
	Mean		1.50E+04	4.69E+04	1.76E+05	6.17E+05
2.5	R1	7.4	1.34E+04	3.98E+04	1.36E+05	4.07E+05	7.8
	R2	7.4	1.34E+04	3.85E+04	1.34E+05	3.94E+05	7.8
	R3	7.4	1.28E+04	4.63E+04	1.31E+05	3.62E+05	7.8
	Mean		1.32E+04	4.15E+04	1.33E+05	3.88E+05
5.0	R1	7.4	1.29E+04	4.59E+04	1.23E+05	2.68E+05	7.8
	R2	7.4	1.27E+04	3.85E+04	1.06E+05	2.61E+05	7.8
	R3	7.4	1.24E+04	3.69E+04	1.03E+05	2.61E+05	7.8
	Mean		1.27E+04	4.04E+04	1.11E+05	2.63E+05
10	R1	7.4	1.26E+04	3.20E+04	8.33E+04	2.27E+05	7.7
	R2	7.4	1.27E+04	2.54E+04	8.22E+04	2.21E+05	7.7
	R3	7.4	1.26E+04	2.17E+04	7.73E+04	2.13E+05	7.7
	Mean		1.26E+04	2.64E+04	8.09E+04	2.20E+05

 

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₃= Replicates 1 to 3

Table 4: Inhibition of Growth Rate and Biomass

Nominal Loading Rate (mg/l)	Area Under Curve at 72h	% Inhibition	Growth Rate (0-72h)	% Inhibition
Control	1.56E+07	-	0.059	-
0.313	1.59E+07	[2]	0.059	0
0.625	1.31E+07	16	0.055	7
1.25	1.19E+07	24	0.052	12
2.5	8.06E+06	48	0.047	20
5.0	6.03E+06	61	0.042	29
10	4.46E+06	71	0.040	32

  [Increase in growth as compared to the controls]

E_bL₅₀(72h) from Figure 2 = 3.5 mg/l loading rate WAF

E_rL₅₀(0 - 72 h) from Figure 2 = 63 mg/l loading rate WAF

Table 5: Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		0.313		0.625
	*	+	*	+	*	+
Height of Medium Column (cm)	13	13	27	27	27	27
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l)
	1.25		2.5		5.0
	*	+	*	+	*	+
Height of Medium Column (cm)	27	27	27	27	27	27
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l) (mg/l)
	10
	*	+
Height of Medium Column (cm)	27	27
Depth of Vortex (cm)	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present

*= Start of mixing period

+= End of mixing period

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated over a 72-Hour period and gave an EbL50 (72 h) value of 3.5 mg/l loading rate WAF and an ErL50 (0-72 h) value of 63 mg/l loading rate WAF. The No Observed Effect Loading rate at 72 hours was 0.313 mg/l loading rate WAF.

* EL = Effective Loading rate
† Value obtained by extrapolation of the growth curve data as no concentration tested resulted in greater than 50% inhibition of growth.

Executive summary:

Introduction

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods

Following preliminary range-finding tests,Scenedesmus subspicatus was exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 0.313, 0.625, 1.25, 2.5, 5.0 and 10 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 determined for each control and treatment group, using a Coulter^®Multisizer II Particle Counter.

Results & Conclusion

Exposure of Scenedesmus subspicatus to the test material gave an E_bL*₅₀(72 h) value of 3.5 mg/l loading rate WAF and an E_rL*₅₀(0 - 72 h) value of 63^†mg/l loading rate WAF (> 10 mg/L WAF, the highest concentration tested). The No Observed Effect Loading rate was 0.313 mg/l loading rate WAF.

*EL = Effective Loading Rate

^†Value obtained by extrapolation of the growth curve data as no concentration tested resulted in greater than 50% inhibition of growth.

Description of key information

EL50 (growth rate) is 63 mg/L WAF loading rate, EC50 (biomass) is 3.5 mg/L WAF loading rate, NOEC (biomass) is 0.313 mg/L WAF loading rate; study performed in line with OECD Guideline 201; Vryenhoef (2002).

Key value for chemical safety assessment

EC50 for freshwater algae:

63 mg/L

EC10 or NOEC for freshwater algae:

0.313 mg/L

Additional information

In a GLP compliant algal inhibition test conducted in line with a standardised guideline, the effect of the test substance on Scenedesmus subspicatus was investigated. Algae were exposed to Water Accommodated Fractions (WAFs) over nominal loading rates of 0.313, 0.625, 1.25, 2.5, 5.0 and 10 mg/L 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 determined for each control and treatment group. Exposure of Scenedesmus subspicatus to the test substance gave an E_bL₅₀ value of 3.5 mg/L loading rate WAF and an E_rL₅₀ value of 63 mg/L loading rate WAF (> 10 mg/L WAF, the highest concentration tested; value obtained by extrapolation of the growth curve data as no concentration tested resulted in greater than 50% inhibition of growth). The No Observed Effect Loading rate was 0.313 mg/L loading rate WAF.