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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:
18 February 1997 - 21 February 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP 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:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
MMBC
Appearance: light brown solid
Storage conditions: in darkness at room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The test substance rapidly hydrolyses to MMBA with a half life of <15 minutes at 25 °C in water. Based on this data, the compound in this study was initially converted to the hydrolysis product MMBA in order to test the compound up to a concentration of 100 mg/L as required in the pertinent guidelines. The method of analysis investigated the concentration of MMBA, however, and all results are expressed as mean measured concentration of test substance equivalents (see Tables 1 and 2).

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Stability of test concentrations
Test concentrations were verified by chemical analysis. Duplicate samples (10 mL) were taken from control and test cultures at 0 and 72 hours (replicates pooled) and sent for analysis. The samples were not filtered to remove algal cells prior to 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:
no
Details on test solutions:
The test substance was added to sterile nutrient medium to give an initial stock solution of 199.2 mg test substance equivalents/L and ultrasonicated for approximately 90 minutes so that the test substance was hydrolysed to MMBA. The pH of this stock solution was then adjusted using 0.7 mL 1N sodium hydroxide to a pH of 7.9. Serial dilutions of this stock solution were prepared with test water to produce a series of solutions exactly twice the concentration of the intended exposure levels. 200 mL of algal pre-culture was mixed with 200 mL of each of these solutions to give the final test series.

CULTURE MEDIUM
Four stock solutions were prepared according to the following table, using reverse osmosis purified/deionised water. Stock solutions were sterilised by autoclaving (solutions 1-3) or by membrane filtration (solution 4) before being stored at +4 °C in the dark.

Aliquots of stock solutions 1 - 3 were further diluted with reverse osmosis purified/deionised water and autoclaved again to produce the working strength nutrient medium. Prior to use, an aliquot of stock solution 4 was added aseptically to the medium via a membrane filter. The pH of the medium after equilibration with air was approximately 8.

Nutrient Concentration Volume of stock solution Final concentration in
in stock solution per litre of final medium test solution

Stock solution 1: macro-nutrients
NH4Cl 1.5 g/L 10mL 15 mg/L
MgCl2.6H2O 1.2 g/L 12 mg/L
CaCl2.2H2O 1.8 g/L 18 mg/L
MgSO4.7H2O 1.5 g/L 15 mg/L
KH2PO4 0.16 g/L 1.6 mg/L


Stock solution 2: Fe-EDTA
FeCl3.6H2O 80 mg/L 1 mL 0.08 mg/L
Na2EDTA.2H2O 100 mg/L 0.1 mg/L


Stock solution 3: trace elements
H3BO3 185 mg/L 1 mL 0.185 mg/L
MnCl2.4H2O 415 mg/L 0.415 mg/L
ZnCl2 3 mg/L 3 x 10^-3 mg/L
CoCI2.6H2O 1.5 mg/L 1.5 x 10^-3 mg/L
CuCI2.2H2O 0.01 mg/L 10^-5 mg/L
Na2MoO4.2H2O 7 mg/L 7 x 10^-3 mg/L


Stock solution 4: NaHCO3
NaHCO3 50 g/L 1 mL 50 mg/L

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST SPECIES
-Strain No.: CCAP 278/4.
-Source: Algal cultures were obtained from the Culture Centre of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK.

PRE-CULTURE
Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (≈ 7000 lux) in an orbital incubator at 23 °C, to give an algal suspension in log phase growth, characterised by a cell density of 3.05 x 10^5cells/mL.
The suspension was diluted using sterile nutrient medium to a cell density of 2.2 x 10^4cells/mL prior to use.

Study design

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

Test conditions

Test temperature:
23 ± 1 °C
pH:
7.2 - 8.3
Nominal and measured concentrations:
Nominal concentrations: 4.58, 9.96, 21.9, 45.8 and 99.6 mg test substance equivalents/L
Mean measured concentrations: 4.91, 10.7, 23.8, 47.5 and 101 mg test substance equivalents/L
Details on test conditions:
EXPOSURE CONDITIONS
Five test concentrations (each in triplicate) were prepared plus 6 replicates of an untreated control. The test concentrations were selected following a range finding study between 0.1 and 100 mg/L.

Culture conditions
Conical flasks (250 mL) each containing 100 mL of test or control culture were loosely stoppered and placed without conscious bias in a Gallenkamp Illuminated Orbital Incubator. The cultures were incubated, without medium renewal, for 72 hours under continuous illumination of approximately 7000 lux provided by 7 x 30 W "universal white" 1 metre fluorescent tubes. The temperature was maintained at 23 ± 1 °C. Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 175 cycles per minute.

MEASUREMENT OF GROWTH
The cell densities of all cultures at initiation and at termination were determined by direct counting using a Coulter Multisizer II particle counter.

EVALUATION OF DATA
The area under each growth curve (cell density versus time) was taken to be an index of growth.
Percentage inhibition of growth at each test concentration was calculated by comparing the area under the test curve with that under the control.

Percentage inhibition of growth values were plotted against test concentration, a line fitted by logistic regression and the EbC50 estimated by interpolation of the fitted curve. The EbC50 ("x" h) is the median effective concentration for inhibition of growth based on a comparison of areas under the growth curves after "x" hours.
The average specific growth rate for each exponentially growing culture was also calculated from the appropriate section of the growth curve.

Percentage reductions in growth rate and the ErC50 value were calculated as for the "area under the curve" data. The ErC50 ("x" - "y" h) is the median effective concentration for inhibition of growth based on a comparison of growth rates from "x" to "y" hours.
The "no-observed effect level" (NOEL) was obtained using Williams' test to compare the percentage inhibition in each treated group with that for the control cultures (Williams' D.A., 1971/72, biometrics 27; 103 - 117 and 28; 519 - 531).
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 9.96 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
67.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % confidence limits 47.9 - 94.5 mg MMBC equivalents/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 99.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Comparison of the measured concentrations of the test substance obtained from test medium samples at a concentration equivalent to the highest test concentration, but without the presence of algal cells, indicated that the presence of algal cells had not affected the stability of the exposure solutions.

The calculated "area under the curve" and "specific growth rate" values are given in Table 3 and are expressed in terms of percentage inhibition by comparing each value with that of the control curve.

Mean cell density of control at 0 h: 1.24 x 10^4 cells/mL
Mean cell density of control at 72 h: 2.29 x 10^6 cells/mL

OBSERVATIONS
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the cultures examined.
No cultures showed any signs of contamination by foreign algal cells or protozoa.
Reported statistics and error estimates:
In order to estimate the concentration at which 50% inhibition of growth occurs, a logistic regression curve (against the logarithm of measured concentration) was fitted to the percentage inhibition values based on the 0 - 72 hour Area Under the Curve. Although the fitted curves were not influenced by the control data, the confidence intervals for the 50% point were adjusted for the control group variability. The data were analysed using both sets of measured concentrations.

It was not possible to obtain a 50% point for the 0 - 72 hour growth rate data since this level of inhibition was not attained.
Williams' test was used to compare the percentage inhibition in each treated group with the baseline (control) values. Bartlett's test for homogeneity of variance was also applied.

DATA HANDLING
The data were entered by hand and analysed using Genstat 5 release 1.3.

Any other information on results incl. tables

Table 1 Test Substance: Measured Concentrations - Mean Values and Percentages of Nominal

Nominal Concentration (mg test substance equivalents/L)

Number of Samples Analysed

Mean Measured Concentration

(mg/L)

 

% Nominal

Control

4.58

9.96

21.9

45.8

99.6

99.6 (No algae)

2

2

2

2

2

2

2

None Detected

4.91

10.

23.8

47.5

101

99.1

-

107

108

109

104

101

99.5

 

Table 2 Summary of Chemical Analysis results

Nominal Concentration (mg test substance equivalents/L)

Measured Concentration

 

 

QC samples % recovery

As MMBA

(mg/L)

As test substance

(mg/L)

As a % of

nominal

0 hours

Control

4.58

9.96

21.9

45.8

99.6

99.6 (No algae)

 

None Detected

4.304

9.342

20.64

41.37

89.38

85.75

 

-

4.803

10.42

23.03

46.17

99.74

95.68

 

-

105

105

105

101

100

96.1

 

-

101

 

98.7

 

 

93.8

72 hours

Control

4.58

9.96

21.9

45.8

99.6

99.6 (No algae)

 

None Detected

4.502

9.914

21.96

43.80

91.05

91.93

 

-

5.023

11.06

24.50

48.87

101.6

102.6

 

-

110

111

112

107

102

103

 

-

105

 

102

 

 

91.8

 

Table 3 Inhibition of Growth

Nominal Concentration (mg test substance equivalents/L)

Mean Measured Concentration (mg test substance equivalents/L)

 

Area under curve at 72 h

 

% Inhibition*

 

Growth Rate (0 - 72 h)

 

% Inhibition*

Control

4.58

9.96

21.9

45.8

99.6

None Detected

4.91

10.7

23.8

47.5

101

3675 

3689

3581

2896

2266

1425

-

0

3

21

38

61

0.0724

0.0737

0.0726

0.0697

0.0670

0.0595

- 

<2>

0

4

7

18

*Percentage inhibition values calculated using non-rounded data

<> Increase when compared to the control

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test substance inhibited the growth of Selenastrum capricornutum, at concentrations tested in excess of 9.96 mg test substance equivalents/L ("No-observed effect level") under the conditions of this test.
The EbC50 (72 h) was 67.3 mg test substance equivalents/L and the ErC50 (0-72 h) was >99.6 mg test substance equivalents/L, the highest concentration tested.
Executive summary:

The toxicity of the test substance to aquatic plants was investigated in the algae Selenastrum capricornutum in accordance with standardised guidelines OECD 201 and EU Method C.3. During the study, algal cultures were exposed to five test concentrations, nominally 4.58, 9.96, 21.9, 45.8, 99.6 mg test substance equivalents/L, plus one untreated control (mean measured concentrations of 4.91, 10.7, 23.8, 47.5 and 101 mg test substance equivalents/L. The compound in this study was initially converted to the hydrolysis product MMBA in order to test the compound up to a concentration of 100 mg/L as required by the guidelines. The method of analysis investigated the concentration of MMBA, however, all results were expressed as the mean measured concentration of test substance equivalents. Cultures were incubated in a Gallenkamp Orbital Incubator under continuous illumination at 23 ± 1 ºC for 72 hours. Cell numbers were counted daily to monitor growth. Under the conditions of the study, a 72 hour EbC50 of 67.3 mg test substance equivalents/L and a 72 hour ErC50 of > 99.6 mg test substance equivalents/L were determined.