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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Feb - 29 Feb 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The pH in the control cultures varied by more than 1.5 units after 96 h.
Qualifier:
according to
Guideline:
EPA OPP 122-2 (Algal Toxicity, Tiers I and II)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples of 100 mL were taken from the stock solutions of control and test cultures at 0 h and pooled replicates at 96 h for analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2-ethylhexyl benzoate is poorly soluble in water and potentially biodegradable. The test substance was initially dispersed in dimethylformamide to give a stock solution of 100 mg/mL. A 500 µL aliquot of this solvent stock was carefully added to the surface of 5 L of autoclaved algal medium to produce an even surface layer without forming a dispersion. This was then left to stir in darkness on a Chiltern magnetic stirrer gently so no visible vortex was seen at the water surface to produce a saturated solution. An aliquot of 8.6 mL of algal pre-culture was mixed with 800 mL of the saturated solution and control media to give the final test series with initial cell densities of 1E04 cells/mL.
- Chemical name of vehicle: Dimethylformamide
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Algal cultures were obtained from the Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK
- Pre-culture: Inoculated from a master culture to give an algal suspension in log phase growth, characterised by a cell density of 9.3E05 cells/mL


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
23 - 24.4 °C
pH:
7.5 - 7.7 at test start
8.0 - 8.1 at test end
Nominal and measured concentrations:
Nominal: Control and 100% saturation
Measured: < LOD and 0.035 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): loosely stoppered
- Size, fill volume: 250 mL, 100 mL
- Aeration: No; shaking on orbital shaker at 120 rpm
- Initial cells density: 1.3E04 cells/mL
- Control end cells density: 3.3E06 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, composition of test medium was equivalent to OECD medium

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: Temperature was recorded daily, pH was measured at test start and at 72 and 96 h.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 3500 - 4500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities of the cultures were determined by direct counting using a Coulter Counter at 0, 24, 48, 72 and 96 h.
- Microscopical inspection of the cells after 96 h.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: A limit study was conducted at 100% saturation, as a saturated solution of the test substance was not expected to show inhibition given that it produced no toxic effects to Daphnia magna.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes, multiplication by a factor of approx. 254
- Observation of abnormalities: Microscopical inspection after 96 h revealed no abnormalities.
- Other: No cultures showed any signs of contamination.
- Inhibition of growth: 7.5% based on biomass and 1.6% based on growth rate at 96 h.
- Effect concentrations exceeding solubility of substance in test medium: Yes

The measured concentrations from the flasks without algal cells indicated that the presence of the algae had not affected the stability of the exposure solutions.

Table 1: Cell densities for control and test cultures

Nominal conc. (% saturation)

Geometric mean meas. conc. (mg/L)

Mean cell densities (cells/mL)

0 h

24 h

48 h

72 h

96 h

Control

< LOD

12,804

30,433

228,420

975,083

3,326,233

100%

0.035

12,881

26,511

208,310

910,983

3,073,267

Description of key information

No adverse effects up to the limit of water solubility (0.4 mg/L measured in aqua dest; 0.035 mg/L in algae test medium) for Selenastrum capricornutum (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.035 mg/L
EC10 or NOEC for freshwater algae:
0.035 mg/L

Additional information

Two GLP-studies with Selenastrum capricornutum according to OECD guideline 201 are available for assessing the toxicity of 2-ethylhexyl benzoate. Six replicate cultures were exposed to both a saturated solution (initial mean measured concentration 0.3045 mg/L) and an untreated control for 96 h (Barrett, 2001). Analysis of the test solutions at the end of the study indicated there had been total degradation of the material over the study period. The result is thus based on the geometric mean measured concentration. Since no significant adverse effects were observed, the NOEC (96 h) and EC50 (96 h) were derived as 0.035 mg/L and > 0.035 mg/L (based on geometric mean measured concentration), respectively. In another study, the same algae species was tested for 72 h (Manson, 2000). This study however did not include analytical confirmation of the test substance concentrations. After 72 h of exposure, no adverse effects on the growth were observed up to 100 mg/L (nominal) which was the highest concentration tested.