2,2-bis(hydroxymethyl)butanoic acid

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:

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

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

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.

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 (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

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

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.

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

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.

Description of key information

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".

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.

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.

Key value for chemical safety assessment

EC50 for freshwater algae:

105 mg/L

EC10 or NOEC for freshwater algae:

48 mg/L

Additional information

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