Registration Dossier

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:
01 Aug - 24 Oct 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 1, 3.2, 10, 32, 100 mg/L
- Sampling method: Samples were taken in duplicate from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 96 h for quantitative analysis.
- Sample storage conditions before analysis: Stored at approximately -20 °C for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test material (100 and 32 mg) were each separately dissolved in culture medium with the aid of ultrasonication for approx. 40 min and the volumes adjusted to 500 mL to give 200 and 64 mg/L stock solutions, respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 20, 6.4 and 2.0 mg/L. An aliquot (250 mL) of each of the stock solutions was separately mixed with algal suspension (250 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures from Culture Collection of Algae and Protozoa (CCAP), Oban, Argyll, Scotland
- Method of cultivation: Cultivated in the laboratory under standard conditions (constant illumination at 21 ± 1 °C).
- Preparation of culture for exposure: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E+04 - 10E+05 cells/mL.

Study design

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

Test conditions

Test temperature:
24 ± 1 °C
pH:
7.6 - 8.2 (0 h)
7.6 - 8.3 (96 h)
Nominal and measured concentrations:
Nominal: control, 1.0, 3.2, 10, 32 and 100 mg/L
Measured (0 h): < LOQ, 0.883, 2.86, 8.97, 29.1, 90.8 mg/L
Measured (96 h): Measured (geometric mean): < LOQ, 0.15, 0.27, 0.48, 0.87, 1.5 mg/L
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: glass, 250 mL, heaspace: 150 mL, fill volume: 100 mL
- Aeration: constantly shaken
- Initial cells density: 1.0E+04 cells/mL
- Control end cells density: 3.51E+06 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionised water
- Culture medium different from test medium: same as test
- Intervals of water quality measurement: regularly

OTHER TEST CONDITIONS
- Sterile test conditions: sterile test medium
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: approx. 7000 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at 0, 24, 47 72 and 96 h and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: control, 0.1, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
zinc chloride

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.70 - 0.82 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Remarks on result:
other: 95% CI: 0.52 - 0.58 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 21 - 28 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Remarks on result:
other: 95% CI: 12 - 14 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 96 h there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/L, however cell debris was observed to be present in the test cultures at 100 mg/L.
- Any stimulation of growth found in any treatment: yes, at 3.2 mg/L (2% stimulation of growth after 72 h)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 96 h test period all control, 1.0, 3.2 and 10 mg/L test cultures were observed to be green dispersions. The 32 mg/L test cultures were observed to be extremely pale green dispersions whilst the 100 mg/L test cultures were observed to be slightly hazy dispersions.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: EC50 (72 h): 0.45 mg/L (95% CI: 0.37 - 0.56 mg/L) based on growth rate
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 and 96 h for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Re-growth was observed to have occurred in the control, 1.0, 3.2 and 10 mg/L test cultures after 48 h, in the 32 mg/L test culture after 96 h and in the 100 mg/L test cultures after 168 h. These results indicate that the test material was algistatic in effect.

Pre-study stability analyses conducted indicated that the test material was unstable in culture medium producing the degradation product benzoic acid. It was therefore considered appropriate to analyse the test samples for both the parent test material and the degradation product. The results are presented in the tables below.

Table 1: Results of the definitive test based on the geometric mean measured concentration after 96 h for completeness

 

Response value

EC50 [mg/L]

95% CI [mg/L]

NOEC [mg/L]

LOEC [mg/L]

96 h

Growth rate

0.91

0.80 - 1.0

0.48

0.87

Yield

0.58

0.55 - 0.62

0.27

0.48

Biomass

0.57

0.53 - 0.60

0.27

0.48

Table 2: Inhibition of growth (0-72 h)

Nominal concentration [mg/L]

Growth rate [cells/mL/h]

0 - 72 h

% inhibition

Control

R1

0.069

-

R2

0.069

R3

0.068

R4

0.069

R5

0.069

R6

0.071

Mean

0.069

SD

0.001

1.0

R1

0.067

3

R2

0.070

[1]

R3

0.070

[1]

Mean

0.069

0

SD

0.002

 

3.2

R1

0.070

[1]

R2

0.071

[3]

R3

0.071

[3]

Mean

0.071

[2]

SD

0.001

 

10

R1

0.064

7

R2

0.066

4

R3

0.067

3

Mean

0.066

5

SD

0.002

 

32

R1

0.028

59

R2

0.017

75

R3

0.016

77

Mean

0.020

70

SD

0.007

 

100

R1

0.004

94

R2

0.006

91

R3

0.007

90

Mean

0.006

92

SD

0.002

 

Values in brackets indicate stimulation of growth.

Analysis of the test preparations at 0 h showed measured test concentrations of the parent test material (vinyl benzoate) to range from 88% to 91% of nominal. Analysis of the test preparations at 96 h showed a decline in measured test concentrations to less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.052 mg/L. The decline in measured concentrations of the test material was in line with the preliminary stability analyses conducted which indicated that test material was unstable in culture medium.

A further decline in measured concentrations was considered to be due to possible adsorption of the test material to the algal cells present. Whilst the recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not preclude long-term adsorption over the test period. Adsorption was not a factor in the preliminary stability analyses since no algal cells were present. Analysis of the test preparations for the degradation product (benzoic acid) at 0 hours showed measured test concentrations to range from 0.052 to 1.3 mg/L. A decline in measured degradant concentrations was observed at 96 h to less than the LOQ of the analytical method employed which was determined to be 0.050 mg/L. Given that benzoic acid was known to be stable this decline was considered to be due to adsorption of the degradation product to the algal cells present.

Given the decline in measured parent test material concentrations, the results have also been determined based on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.026 mg/L) was used to enable calculation of the geometric mean measured concentration.

Table 3: Analytical results of the test item

Sample

Nominal concentration [mg/L]

Measured concentration [mg/L]

% of nominal

0 h

Control

< LOQ

-

1.0

0.883

88

3.2

2.86

89

10

8.97

90

32

29.1

91

100

90.8

91

96 h

Control

< LOQ

-

1.0

< LOQ

-

3.2

< LOQ

-

10

< LOQ

-

32

< LOQ

-

100

< LOQ

-

 

Table 4: Analytical results of benzoic acid

Sample

Nominal concentration [mg/L]

Measured concentration [mg/L]

0 h

Control

< LOQ

1.0

0.052

3.2

0.11

10

0.15

32

0.47

100

1.28

96 h

Control

< LOQ

1.0

< LOQ

3.2

< LOQ

10

< LOQ

32

< LOQ

100

< LOQ

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes