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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-01-1997 to 27-01-1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study; no data on mean CV for section-by-section specific growth rate and average specific growth rates during the whole test period in replicate control cultures

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2-bis(hydroxymethyl)butanoic acid
EC Number:
424-090-1
EC Name:
2,2-bis(hydroxymethyl)butanoic acid
Cas Number:
10097-02-6
Molecular formula:
C6H12O4
IUPAC Name:
2,2-bis(hydroxymethyl)butanoic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): DMBA
- Physical state: White powder
- Analytical purity: 99.0%
- Lot/batch No.: 5JO1
- Expiration date of the lot/batch: 09 April 1997
- Storage condition of test material: In darkness at room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling: Duplicate samples (50 mL) were taken from control and test concentrations (buffered and unbuffered) at 0 and 72 hours (replicates pooled) for analysis. Additional samples were also taken from flasks containing the test substance at a concentration equivalent to the highest exposure concentration but with no algae present, at 0 and 72 hours (replicates pooled), in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was dissolved in sterile nutrient medium to give an initial stock solution of 200 mg/L. This stock solution was further diluted with sterile nutrient medium to produce a series of solutions exactly twice the concentration of the intended exposure levels. 500 mL of algal pre-culture was mixed with 500 mL of each of these solutions to give the final test series.
- Controls: The control group was maintained under identical conditions and contained dilution medium only.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Centre of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (ca.4000 lux) in an orbital incubator at 24 ± 1 °C, to give an algal suspension in log phase growth, characterised by a cell density of 5.3 x 10^6 cells/mL. The suspension was diluted using sterile nutrient medium to a cell density of 2.0 x 10^4 cells/mL prior to use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
No data provided
Test temperature:
23 °C
pH:
4.5-7.9 (unbuffered solutions) and 7.0-7.8 (buffered solutions)
Dissolved oxygen:
No data provided
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: 0, 4.6, 10, 22, 46 and 100 mg/L
- Mean measured concentrations: 0, 4.9, 10, 23, 48 and 99 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks (250 mL) each containing 100 mL of test or control culture were loosely capped with foil and placed without conscious bias in a Gallenkamp Illuminated Orbital Incubator.
- Aeration: Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 120 cycles/min.
- Initial cells density: 10000 cells/mL
- Control end cells density: 1.3 x 10^6 cells/mL
- No. of vessels per concentration (replicates): Triplicate
- No. of vessels per blank control (replicates): Triplicate
- An additional two concentrations (equivalent to the highest concentrations tested) and control series were also prepared and buffered by the addition of potassium dihydrogen orthophosphate.

GROWTH MEDIUM
- Standard medium used: Yes; Sterile nutrient medium as recommended in EU Method C.3.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile nutrient medium prepared using reverse osmosis purified/deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Yes; an additional two concentrations (equivalent to the highest concentrations tested) and control series were prepared and, before inoculation with algal cells, buffered by the addition of potassium dihydrogen orthophosphate (1 mL). Buffered test solutions were adjusted to measure pH 7.3 using 0.01 N NaOH.
- Photoperiod: Continuous illumination provided by 7 x 30 W ‘universal white’ 1 m fluorescent tubes.
- Light intensity and quality: Approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell density was determined by direct counting using a Coulter® Multisizer II particle counter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: In the range-finding study, DMBA was found to be inhibitory at concentrations above 10 mg/L with approximately 90 % inhibition evident for the 100 mg/L solution. At this time, it was noted that DMBA had reduced the pH of the test solution from 8.0 to 5.1 at 100 mg/L and it was considered that this shift in pH might be in part responsible for the inhibition.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
30 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % CL = 25-36 mg/L; non-buffered solution
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
42 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL = 41-44 mg/L; non-buffered solution
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
81 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: buffered solution
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
105 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: buffered solution
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): At 72 hours, no abnormalities were detected in any of the cultures exposed at ≤ 46 mg/L. Cells in the cultures exposed at 100 mg/L were paler than those in the control cultures.
- Other: No cultures showed any signs of contamination by foreign algal cells or protozoa.
- NOEC (72 h) = 23 mg/L (non-buffered solution) and 48 mg/L (buffered solution)
- Refer table 6.1.5/2 and figures 6.1.5/1 and 6.1.5/2 for more data
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- Effective concentrations were estimated using logistic regression curves.
- Due to the limited amount of data available for the buffered series, the lower and upper asymptotes of the fitted curves were fixed at the 0 and 100 % inhibition levels, respectively. Williams' test (Williams, 1971, 1972) was used to compare the percentage inhibition in each treated group with the baseline (control) values. Bartlett's test for homogeneity of variance (Bartlett, 1937) was also applied.
- For the unbuffered series data, Bartlett's test indicated significant heterogeneity of variance, due to a consistent decrease in variability with increasing concentration. The non-parametric equivalent of Williams' test (Shirley, 1977, Williams, 1986) was therefore applied to these data and the results reported in place of those from the Williams' tests.

Any other information on results incl. tables

Table 6.1.5/2: Inhibition of growth

Nominal concentration (mg/L)

Mean measured concentration (mg/L)

Area under curve (72 h)

% Inhibition*

Growth rate (0-72 h)

% Inhibition*

Control

ND

2338

-

0.06709

-

4.6

4.9

2192

6

0.06699

0

10

10

2291

2

0.06750

0

22

23

1962

16

0.06482

3

46

46

205

91

0.02796

58

100

99

85

96

0.01377

79

Buffered concentrations

Control

ND

2201

-

0.06625

-

46

48

2144

3

0.06558

1

100

100

429

81

0.03816

42

* Percentage inhibition values calculated using non-rounded data

ND None detected

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Remarks:
control cultures (0-72 h): cell concentration increased by approximately 100 folds; no data on mean CV for section-by-section specific growth rate and average specific growth rates during the whole test period in replicate control cultures
Conclusions:
Under the test conditions, DMBA inhibited the growth of algae at concentrations tested in excess of 23 mg/L. The following values were derived from the data: EbC50 (72 h): 30 mg/L; ErC50 (0-72 h): 42 mg/L and No Observed Effect Concentration (NOEC): 23 mg/L.
The following values were derived using data from treatment solutions buffered to remove any pH related effects: EbC50 (72 h): 81 mg/L; ErC50 (0-72 h): 105 mg/L and No Observed Effect Concentration (NOEC): 48 mg/L.
All results were based on mean measured concentrations, which ranged from 99- 106% in this study.
It was considered by the author that the inhibition observed in non-buffered solutions was partly due to the low pH of test solutions and was not wholly attributable to the direct toxicity of DMBA. For this reason, EC50 and NOEC values determined from buffered cultures were believed to be more indicative of the intrinsic biological activity of DMBA than the equivalent values derived from unbuffered test cultures.
Executive summary:

Introduction

A study was conducted to assess the inhibitory effect of DMBA on the growth of the unicellular green alga Selenastrum capricornutum, Strain No. CCAP 278/4. The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 "Algal Inhibition Test" and the OECD Guideline for Testing of Chemicals No. 201 "Alga, Growth Inhibition Test".

Methods

Triplicate algal cultures were exposed to five test concentrations, nominally 4.6, 10, 22, 46 and 100 mg/L, plus one untreated control. Additional pH buffered test concentrations were prepared for the two highest concentrations where a substantial reduction in pH was observed at the start of the study.

An additional control series containing the pH buffer was also prepared. All the cultures were incubated in a Gallenkamp Orbital Incubator under continuous illumination at 23 °C for 72 hours. Cell numbers were counted daily to monitor growth.

Results

Under the test conditions, DMBA inhibited the growth of algae at concentrations tested in excess of 23 mg/L. The following values were derived from the data: EbC50 (72 h): 30 mg/L; ErC50 (0-72 h): 42 mg/L and No Observed Effect Concentration (NOEC): 23 mg/L.

The following values were derived using data from treatment solutions buffered to remove any pH related effects: EbC50 (72 h): 81 mg/L; ErC50 (0-72 h): 105 mg/L and No Observed Effect Concentration (NOEC): 48 mg/L.

All results were based on mean measured concentrations, which ranged from 99- 106% in this study.

It was considered by the author that the inhibition observed in non-buffered solutions was partly due to the low pH of test solutions and was not wholly attributable to the direct toxicity of DMBA. For this reason, EC50 and NOEC values determined from buffered cultures were believed to be more indicative of the intrinsic biological activity of DMBA than the equivalent values derived from unbuffered test cultures.

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