4-methylstyrene

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 May 19, 2000 to September 04, 2001

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:

yes

Remarks:

Conducted in sealed vessels to avoid volatilisation

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

Although the test substance is stable in water it is volatile, therefore both the range finding and definitive study were conducted in sealed vessels. The study design used was static. Test solutions were sampled at the start and end of the study.

Vehicle:

no

Details on test solutions:

Water used for acclimatization of the test organisms and all toxicity testing was sterile freshwater AAP medium at a pH of .75. A series of test solutions was prepared by bringing 0.0010, 0.0020, 0.0040, 0.0081 and 0.016 g of the test substance to 1,000 mL dilution water in a sealed amber glass bottles. The solutions were mixed on magnetic stirrers for approximately four hours. The solutions were allowed to settle for approximately one hour and a portion for each solution was transferred into a 1.4 L beaker through a spout at the bottom of the mixing vessels. A 1.4 L portion of dilution water was also transferred to a glass beaker to serve as a control. Water quality measurements were made and each solution was inoculated with approximately 10,000 algal cells/mL.
Solutions were subdivided into 11 clear glass 40 mL vials for each treatment (the control was subdivided into 20 replicates) and the vials, which were filled to capacity to eliminate any head space, were sealed with Teflon-lined caps.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: UTEX 1648
- Source: University of Texas culture collection of algae
- Age of inoculum (at test initiation): eight days
- Method of cultivation: in identical media to that used in the study.
- Acclimation period: fourteen days
- Culturing media and conditions: identical to the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 +/- 2°C

pH:

Target pH 7.5 initial starting pH in the definitive study 7.3 - 7.6. Final pH after 72 h ranged from 8.5- 8.8 in untreated control samples to 7.2-7.4 in the 8.4 mg/L vials containing the test substance.

Nominal and measured concentrations:

Nominal concentrations: 0 (untreated control), 1.0, 2.0, 4.0, 8.1 and 16 mg/L. Mean measured concentrations 0 (untreated control), 0.41, 0.53, 1.6, 3.7 and 8.4 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 ml glass vials, closed, no headspace
- Innoculation level: 10,000 cells/ml
- Source/preparation of dilution water: Local source -filtered and sterilised
- Intervals of water quality measurement: 6 months
- Adjustment of pH: Yes 7.5
- Test concentrations: 0.1, 1.0, 5.0, 10.0,100.0 mg/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Exponential growth in the control (for algal test): yes 2290,000 cells/ml in UTC after 72 h

Mean measured concentrations were: 0.41, 0.53, 1.6, 3.7 and 8.4 mg/L. The mean measured concentrations ranged from 27 to 53 % of the nominal concentrations of the solutions prepared in the beginning of toxicity test. However, the final measured concentrations were 78 to 88 % of the initial measured concentrations, indicating that once aqueous solutions of the test substance were sealed into the test vessels with the algae, concentrations remained relatively constant. Insoluble material was not observed at any tested concentration during the test.

At the conclusion of the definitive toxicity study a further test on the 8.4 mg/L concentration which resulted in complete inhibition of growth was diluted in fresh media and cultured for a further 192 h. The results of the study showed that the effects of the test substance was algistatic rather than algicidal. This has important implications in the event of major accidental environmental releases in that algal growth will recommence once concentrations in surface waters decline below the algistatic level.

Reported statistics and error estimates:

EC50 calculated using non linear regression analysis (Bruce and Versteeg 1992). NOEC calculated using TOXSTAT 3.3 Bonaferroni's test.

Information on the nominal and measured concentration in the definitive study is shown in table below.

Nominal conc mg/L	Measured concentration mg/L
	0 hrs	% of nominal	72 hrs	% of nominal
0	ND	ND	ND	ND
1.0	0.46	46	0.36	36
2.0	0.56	28	0.49	25
4.0	1.7	43	1.4	35
8.1	4.0	49	3.3	41
16.0	9.3	58	7.5	47
Laboratory control sample.
4.0	2.2 2.0	55 50	2.3 2.3	58 58
Stability blank
16	9.0	56	8.4	53

The results of algal growth assessments based on cell counts are shown in the table below.

Measured conc mg/L	Cells per ml (mean of 6 replicates) in thousands
	0 hrs	24 hrs	48 hrs	72 hrs
0	10	23	52	229
0.41	10	22	59	217
0.53	10	22	50	213
1.6	10	18	31	184
3.7	10	13	19	61
8.4	10	10	10	16

The results of the algal growth assessments shown as a percentage of the untreated control.

Measured conc mg/L	% cell growth in comparison to the control
	24 hrs	48 hrs	72 hrs
0.41	94	109	100
0.53	94	100	98
1.6	69	71	93
3.7	31	38	58
8.4	0	0	16

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h mean measured EC50 was established at 2.6, 2.3 and 4.3 mg/L based on cell number, area under the growth curve and growth rate, respectively. The corresponding NOECs were 1.6, 0.53 and 1.6 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the test substance to the green algae Selenastrum capricornutum according to OECD Guideline 201, in compliance with GLP. The test was performed under static conditions in sealed containers from which all air space had been removed to minimize the loss of the volatile test substance. The study was run with five nominal concentrations of 0, 1.0, 2.0, 4.0, 8.1 and 16 mg/L. The mean measured concentrations ranged from 27 to 53% of the nominal values but analytical verification showed that the test substance was stable in the algal growth media. Under the study conditions, the 72 h mean measured EC50 was established at 2.6, 2.3 and 4.3 mg/L based on cell number, area under the growth curve and growth rate, respectively. The corresponding NOECs were 1.6, 0.53 and 1.6 mg/L (Wyskiel, 2001).

Key value for chemical safety assessment

EC50 for freshwater algae:

4.3 mg/L

EC10 or NOEC for freshwater algae:

1.6 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to the green algae Selenastrum capricornutum according to OECD Guideline 201, in compliance with GLP. The test was performed under static conditions in sealed containers from which all air space had been removed to minimize the loss of the volatile test substance. The study was run with five nominal concentrations of 0, 1.0, 2.0, 4.0, 8.1 and 16 mg/L. The mean measured concentrations ranged from 27 to 53% of the nominal values but analytical verification showed that the test substance was stable in the algal growth media. Under the study conditions, the 72 h mean measured EC50 was established at 2.6, 2.3 and 4.3 mg/L based on cell number, area under the growth curve and growth rate, respectively. The corresponding NOECs were 1.6, 0.53 and 1.6 mg/L (Wyskiel, 2001).