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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:
29 Jan 2016 - 10 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Except for the following: the characterisation of the test item was conducted in a GMP environment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: off-white powder
- Storage condition of test material: At room temperature
Specific details on test material used for the study:
Solubility in water: 5.4 mg/L (20°C)
Stability in water: yes

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: two sets of 100 % of SS prepared at 100 mg/L
- Sampling method: singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2.0 mL
Storage: Samples were stored in a freezer until analysis
The filter containing the undissolved residue was kept for possible analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
Method of preparation of test solution: Preparation of test solutions started with the highest test concentration of 100 mg/L applying 2 days of magnetic stirring to ensure maximum dissolution of the test item in the test medium. The resulting dispersion was filtered through a 0.45 μm membrane filter (Whatman; RC55) to remove the undissolved test item, which resulted in a clear and colourless saturated solution (SS). The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. Note that weighing and formulation of test solutions were performed protected from light as much as possible and that the control group received a similar treatment (i.e. filtration).
- Controls: test medium without test item or other aditives
- Evidence of undissolved material: no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: alga
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Method of cultivation: algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL.
- Culturing media and conditions: yes

Study design

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

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
Between 22 and 23°C throughout the test
pH:
Between 8.0 and 8.2 throughout the test
Nominal and measured concentrations:
Nominal: 1.0, 10 and 100 mg/L
Measured: average measured concentrations were 2.66, 1.44 and 2.19 mg/L at t=0, t=24 and t=72 h, respectively
The measured concentrations remained fairly stable during the study and consequently a mean measured exposure concentration was based on the results of the duplicate samples analysed at the three time points.Mean concentration at t=0-72 h: 2.1 mg/L
Details on test conditions:
TEST SYSTEM:
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 161.9 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each intermediate concentration, 1 replicate of each test concentration without algae for turbidity correction
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard growth medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture (stock) medium different from test medium: yes (M1)
- Pre-culture medium different form test medium: no

OTHER TEST CONDITIONS:
- Adjustment of pH: no
- Sterile test conditions: not mentioned
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 90 to 96 μE/m2/s.

EFFECT PARAMETERS MEASURED: growth rate and yield after 72 hours. In addition, pH was measured at the beginning and at the end of the test, temperature of the medium was continuously measured in a temperature control vessel and the appearance of cells was measured at the end of the final test (including observations for any abnormal appearance of the algae).

- Determination of cell concentrations: cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions. At t=0, t=24, t=48 and t=72 for control and the limit concentration, at t=72 for intermediate concentrations.
- Other: In order to correct for turbidity one control per concentration, without algae, was measured at each time interval.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study concentations: 1.0, 10 and 100% of a SS prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: no, a combined limit/range-finding test was used and results were not used to determine the conditions for the final study.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (December 2015)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Details on results:
.- Exponential growth in the control (for algal test): yes
- No biological, behavioural or other abnormalities were observed.
- Effect concentrations exceeding solubility of substance in test medium: yes, effect concentrations > 2.1 mg/L

pH and temperature maintained within the limits prescribed by the study plan during the study.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.5 mg/L, 95%-CI (1.4, 1.6 mg/L)
- EyC50: 0.51 mg/L, 95%-CI (0.49, 0.53 mg/L)

Any other information on results incl. tables

Table 1       Concentrations of the test item in test medium – reserve samples

Time of sampling
[hours]

Percentage of SS2
[%]

Analysed concentration
[mg/L]

Relative to
initial
[%]

 

 

 

 

0

0

n.d.

 

 

100

2.18

 

 

100

 2.213

 

 

 

 

 

24

0

n.d.

n.a.

 

100

1.33

61

 

100

 1.333

60

 

 

 

 

72

0

n.d.

n.a.

 

100

1.84

84

 

100

 1.693

76

 

 

 

 

1          Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2          Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

3          Without algae.

n.d.    Not detected.

n.a.     Not applicable.

 

 

Table 2            Percentage inhibition of growth rate (total test period)

Broomdex

%SS

Mean

Std. Dev.

n

%Inhibition

Control

1.694

0.0328

6

1.0

1.680

0.0149

3

0.8

10

1.670

0.0089

3

1.4

100 (2.1 mg/L)

1.688

0.0400

6

0.4

( ) Mean measured concentration

Table 3            Percentage inhibition of yield(total test period)

Broomdex

%SS

Mean

Std. Dev.

n

%Inhibition

Control

160.9

16.22

6

1.0

153.7

6.87

3

4.5

10

148.9

4.02

3

7.5

100 (2.1 mg/L)

158.0

18.74

6

1.8

( ) Mean measured concentration

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1) Control cell density increased > 16-fold. 2) Mean coefficient of variation for section-by-section specific growth rates in the control <35%. 3) CV of average specific growth rates during the whole test period in replicate control cultures <7%
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of Broomdex tested. Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50)
exceeded 2.1 mg/L, being considered the mean measured concentration in a SS prepared at 100 mg/L. The 72h-NOEC for growth rate inhibition and yield inhibition was 2.1 mg/L, being considered the mean measured concentration in a SS prepared at 100 mg/L.
Due to the very low solubility of Broomdex in water, concentration levels that might be toxic for algae could not be reached.