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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:
10-18 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 201 without any deviation
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: Clear colorless viscous liquid (after heating)
- Lot/batch No.: 11GP0077
- Expiration date of the lot/batch: 1 April 2014
- Storage condition of test material: Room temperature in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples were taken from the uninoculated control and 100 mg/L loading rate WAF test group at 0 and 72 h for Total Organic Carbon (TOC) analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was heated to 50 °C prior to use. An amount of test item (200 mg) was weighed and added to the surface of 2 L of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 h and the mixture allowed to stand for 1 h. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. An aliquot (1 L) of the WAF was inoculated with algal suspension (6.3 mL) to give the required test concentration of 100 mg/L loading rate WAF.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland

ACCLIMATION
- Culturing media and conditions (same as test or not): Yes, same as test

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No data

Test conditions

Hardness:
No data
Test temperature:
24 ± 1 °C
pH:
7.3-7.8
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
- Range-finding test: 10 and 100 mg/L loading rate WAF (nominal)
- Definitive test: 100 mg/L loading rate WAF (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks containing 100 mL of test preparation and plugged with polyurethane foam bungs
- Initial nominal cell density: 5000 cells/mL
- Control end cells density: 693000 cells/mL
- No. of vessels per concentration (replicates): Six
- No. of vessels per control (replicates): Six

GROWTH MEDIUM
- Standard medium used: Yes, AAP-medium (US. EPA)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionized water (Elga Optima 15+)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380-730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 h and the cell densities were determined using a Coulter®Multisizer Particle Counter.
- pH was measured at initiation of the test and after 72 h of exposure using a WTW pH 320 pH meter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10 and 100 mg/L loading rate WAF
- Results used to determine the conditions for the definitive study: In the range-finding test, no significant effect on growth rate was noted at 10 and 100 mg/L loading rate WAF. Based on this information a single loading rate of six replicates of 100 mg/L, using a stirring period of 23 h followed by a 1 h standing period, was selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (non-concurrent): 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
other: ErL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EyL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
other: growth rate and yield
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No abnormalities were detected in any of the control or test cultures.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72 h test period all control and test cultures were observed to be green dispersions.
- Aggregation of algal cells: Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.
- For more details, refer tables 6.1.5/1, 6.1.5/2 and 6.1.5/3
Results with reference substance (positive control):
- ErC50 (0-72 h): 1.4 mg/L (1.2-1.7 mg/L)
- EyC50 (0-72 h): 0.59 mg/L (0.53-0.65 mg/L)
- No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
- No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
- Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
- Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
Reported statistics and error estimates:
- A Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 h for the control and the 100 mg/L loading rate 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

- Validation of Mixing Period: Pre-study work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 h.

- Chemical analysis results: Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 h which showed measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/L were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

 

Table 6.1.5/1: Cell densities and pH values in the definitive test

Nominal Concentration
(mg/L)

pH

Cell Densities* (cells per mL)

pH

0 h

0 h

24 h

48 h

72h

72h

Control

R1
R2
R3
R4
R5
R6
Mean

7.3

6.96E+03
6.77E+03
6.13E+03
6.03E+03
5.94E+03
7.25E+03
6.51E+03

2.48E+04
1.69E+04
2.18E+04
1.88E+04
2.38E+04
1.64E+04
2.04E+04

1.29E+05
7.22E+04
1.26E+05
1.06E+05
1.30E+05
1.10E+05
1.12E+05

8.12E+05
5.06E+05
7.94E+05
6.49E+05
7.02E+05
6.96E+05
6.93E+05

7.8

100

R1
R2
R3
R4
R5
R6
Mean

7.3

5.89E+03
5.23E+03
5.28E+03
5.79E+03
5.20E+03
6.02E+03
5.57E+03

2.06E+04
1.65E+04
2.19E+04
1.83E+04
1.90E+04
1.59E+04
1.87E+04

1.28E+05
8.88E+04
1.07E+05
1.18E+05
1.16E+05
7.87E+04
1.06E+05

7.55E+05
5.78E+05
6.08E+05
6.42E+05
6.88E+05
5.04E+05
6.29E+05

7.8

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1-R6 = Replicates 1 to 6

 

Table 6.1.5/2: Daily specific growth rates for the control cultures in the definitive test

Daily Specific Growth Rate (cells/mL/h)

Day 0-1

Day 1-2

Day 2-3

Control

R1

0.067

0.069

0.077

R2

0.051

0.061

0.081

R3

0.061

0.073

0.077

R4

0.055

0.072

0.076

R5

0.065

0.071

0.070

R6

0.049

0.079

0.077

Mean

0.058

0.071

0.076

Table 6.1.5/3: Inhibition of growth rate and yield in the definitive test

Nominal Loading Rate
(mg/L)

Growth Rate
(cells/mL/h)

Yield
(cells/mL)

0-72 h

% Inhibition*

0-72 h

% Inhibition*

Control

R1
R2
R3
R4
R5
R6
Mean
SD

0.071
0.064
0.070
0.068
0.069
0.069
0.069
0.002

-

 

 

 

8.05E+05
4.99E+05
7.88E+05
6.43E+05
6.96E+05
6.89E+05
6.87E+05
1.11E+05

-

100

R1
R2
R3
R4
R5
R6
Mean
SD

0.070
0.066
0.067
0.067
0.068
0.064
0.067
0.002

[1]
4
3
3
1
7
3

7.49E+05
5.73E+05
6.03E+05
6.36E+05
6.83E+05
4.98E+05
6.24E+05
8.73E+04

9

* In accordance with the OECD test guideline only the mean value for yield is calculated

SD = Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
control cultures (0-72 h): cell concentration increased by 106 folds; mean CV for section by section specific growth rate was 14 %; CV for average specific growth rate was 3 %
Conclusions:
Under the test conditions, the 72 h ErL50 and EyL50 for test item is greater than 100 mg/L loading rate WAF in green algae (Pseudokirchneriella subcapitata). The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
Executive summary:

In an algal toxicity study performed in accordance with OECD guideline 201 and GLP, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item,at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 h, under constant illumination and shaking at 24 ± 1 °C. Measurement of biomass and growth inhibition was done by determination of cell densities at 0, 24, 48 and 72 h using electronic particle counter. A preliminary range-finding test was also conducted during which no effect on algal growth was observed at 10 and 100 mg/L loading rate WAF.

In the main test, after 72 h, cell concentration in the control culture increased by 106 folds; mean CV for section by section specific growth rate was 14 and CV for average specific growth rate was 3 %. Hence, it met the validity criteria for the control vessels. The ErL50 (effective loading rate) and EyL50 at 72 h were determined to be >100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg /L. Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 h which showed measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/L were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Under the test conditions, the 72 h ErL50 and EyL50 for test material is greater than 100 mg/L loading rate WAF in green algae (Pseudokirchneriella subcapitata). The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.