Benzene, poly(propene) derivatives

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(see below)

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several regulatory authorities (ECETOC, OECD) is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/L) of test material in dechlorinated tap water for 24 hours and then removing the undissolved test material by filtration through a pre-conditioned filter (0.2 µm) to give a 100% v/v saturated solution.

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): AL305B
- Physical state: clear, colourless, slightly viscous liquid
- Lot/batch No.: TS07003
- Analytical purity: 100%
- Expiration date of the lot/batch: 24 May 2007
- Storage condition of test material: room temperature, in the dark

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

An amount of test material (1100 mg) was added to culture medium and the volume adjusted to 11 litres to give an initial test material dispersion of 100 mg/L. This was stirred using a propeller stirrer set at approximately 1500 rpm for 24 hours at 21ºC. After stirring the undissolved test material was removed by filtration through a 0.2 µm Sartopore filter (approximately 1 litre discarded to pre-condition the filter) to give the 100% v/v saturated solution from which serial dilutions were made to give 10 and 1.0 % v/v saturated solutions. An aliquot (500 mL) of each saturated solution was inoculated with algal suspension (3.6 mL) to give the required test concentrations of 1.0, 10 and 100 % v/v saturated solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

Liquid cultures of P. subcapitata were obtained from the Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 ºC.

Prior to the start of the test, sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under contstant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 ± 1 ºC until the algal cell density was approximately 10E+4 - 10E+5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

Not stated

Test temperature:

24 ± 1 ºC

pH:

7.9-8.1

Dissolved oxygen:

Not measured

Salinity:

Not stated

Nominal and measured concentrations:

100% v/v saturated solution.

Details on test conditions:

Six 250 mL glass conical flasks each containing 100 mL of test preparation were used for the control and 100% v/v saturated solution treatment groups. 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 4.42 x 10E+5 cells/mL. Inoculation of 2 litres of test medium with 45 mL of this algal suspension gave an initial nominal cell density of 1 x 10E+4 cells/mL and had no significant effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm, white lighting and consistently shaken at approx 150 rpm for 72 hours.

Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter Multisize Particle Counter.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v

Basis for effect:

growth rate

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v

Basis for effect:

other: yield

Effect concentrations 3

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v

Basis for effect:

biomass

Effect concentrations 4

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v

Basis for effect:

growth rate

Effect concentrations 5

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v

Basis for effect:

other: yield

Effect concentrations 6

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v

Basis for effect:

biomass

Details on results:

Growth rate, yield and biomass integral were not affected by the presence of a 100% v/v saturated solution of the test material over the 96-hour exposure period.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out usign a Student's t-test incorporating Bartlett's test for homogenieity of variance (Sokal and Rohlf, 1981). At 72 hours, the 100% v/v saturation solution test group was shown to be significantly different (P <0.05) from the control group but at 96 hours there were no statistically significant differences (p ≥ 0.05) between the control and 100% v/v saturated solution test group. Examination of the growth curves for the control and test cultues indicated no signficant biological differences in growth rate and therefore the NOEC based on grwoth rate was considered to be 100% v/v saturated solution.

Statistical analysis of the yield data showed that at 72 hours the 100% v/v saturated soolution test group was significantly different (p <0.05) from the control group. However, there were no statistically significant differences ( p ≥ 0.05) at 96 hours. Examination of the growth curves and test cultures indicated no significant biological differences in growth between the control and test cultures and the NOEC based on yield was considered to be 100% v/v saturated solution.

Statistical analysis of biomass integral data showed no statistically signficant differences (p ≥ 0.05) between the control and 100% v/v saturated solution at 72 or 96 hours and therefore the NOEC based on biomass integral was 100% v/v saturated solution.

Any other information on results incl. tables

Table 1. Inhibition of growth rate, yield and biomass integral after 72 hours in the definitive test

Nominal concentration (% v/v saturated solution)		Growth rate (cells/mL/hour)		Yield (cells/mL)		Biomass integral
		0-72 h	% inhibition	0-72 h	% inhibition	0-72 h	% inhibition
Control	R1	0.069	-	1.44E+06	-	2.39E+07	-
	R2	0.069		1.40E+06		2.33E+07
	R3	0.071		1.64E+06		2.90E+07
	R4	0.068		1.31E+06		2.18E+07
	R5	0.070		1.53E+06		2.65E+07
	R6	0.069		1.39E+06		2.48E+07
	Mean	0.069		1.45E+06		2.49E+07
	SD	0.001		1.19E+06		2.57E+06
100	R1	0.066	4	1.17E+06		2.24E+07	10
	R2	0.066	4	1.12E+06		2.00E+07	20
	R3	0.068	1	1.37E+06		2.51E+07	[1]
	R4	0.069	0	1.39E+06		2.54E+07	[2]
	R5	0.067	3	1.23E+06		2.28E+07	8
	R6	0.067	3	1.24E+06		2.28E+07	8
	Mean	0.067	3	1.25E+06	14	2.31E+07	7
	SD	0.001		1.06E+05		1.98E+06

Table 2. Inhibition of growth rate, yield and biomass integral after 96 hours in the defintive test

Nominal concentration (% v/v saturated solution)		Growth rate (cells/mL/hour)		Yield (cells/mL)		Biomass integral
		0-72 h	% inhibition	0-72 h	% inhibition	0-72 h	% inhibition
Control	R1	0.058	-	2.65E+06	-	7.31E+07	-
	R2	0.058		2.55E+06		7.07E+07
	R3	0.06		3.07E+06		8.56E+07
	R4	0.058		2.70E+06		6.98E+07
	R5	0.059		2.88E+06		7.94E+07
	R6	0.065*		2.77E+06		1.01E+08*
	Mean	0.059		4.96E+06*		7.57E+07
	SD	0.001		2.05E+05		6.68E+06
100	R1	0.06	[2]	3.13E+06		7.40E+07	2
	R2	0.059	0	2.83E+06		6.74E+07	11
	R3	0.06	[2]	3.26E+06		8.06E+07	[6]
	R4	0.06	[2]	3.18E+06		8.02E+07	[6]
	R5	0.059	0	2.93E+06		7.28E+07	4
	R6	0.06	[2]	3.05E+06		7.42E+07	2
	Mean	0.06	[1]	3.06E+06	[11]	7.49E+07	1
	SD	0.001		1.62E+05		6.97E+06

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Validity criteria fulfilled as specified above.

Conclusions:

The effect of AL305B on P. subcapitata resulted in EC50 values of >100% v/v saturated solution and a NOEC of 100% v/v saturated solution. The test substance is not considered to be classified for effects on algae.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata.The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), US CFR Title 40 Part 797 Section 1050 and the US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400.

 

Methods.

Following a preliminary range-fmding test, Pseudokirchneriella subcapitata (six replicate flasks) was exposed to a saturated solution of the test material for 96 hours, under constant illumination and shaking at a temperature of 24 ± 1ºC. The test material solution was prepared by stirring an excess (100 mg/l) of test material via propeller stirrer in culture medium at approximately 1500 rpm at a temperature of 21°C for 24 hours prior to removing any undissolved test material by filtration (0.2 µm Sartopore filter, first approximate 1 litre discarded in order to pre-condition the filter) 10 produce a saturated solution. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

Results.

Exposure of Pseudokirchneriella subcapitata to the test material gave EC50 values of greater than 100% v/v saturated solution and correspondingly the No Observed Effect Concentration was 100% v/v saturated solution. Analysis of the test preparations at 0 and 96 hours showed the measured concentrations to be less than the limit of quantitation of the analytical method. This does not infer that no test material was in solution but that the dissolved concentration (i.e. bioavailable to the test organisms) was below the limit of quantitation which was assessed down to 0.0038 mg/l.

This study showed that there were no toxic effects at saturation.