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:
23 Apr - 03 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006, Annex 5 corrected 28 Jul 2011
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Remarks:
UPLC
Details on sampling:
- Concentrations: Control and all test concentrations at 0, 24 and 72 h
- Sampling method: 2.0 mL from the approximate centre of the test vessels. At the end of the test, the replicates with algae were pooled at each concentration before sampling. In addition, reserve samples of 2.0 mL were taken from all test solutions.
- Sample storage conditions before analysis: In a freezer (≤ - 15 °C)

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Test item: The test item was a colourless to pale yellow liquid with a purity of 99.57% which was completely soluble in test medium at the tested concentrations. No correction was made for the purity/composition of the test item.
- Method: First, the highest test concentration of 100 mg/L was prepared by application of 15 min magnetic stirring for acceleration of the dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of this limit concentration in test medium.
- Differential loading: No
- Controls: Test medium without test item
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum: 3 d before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E-04 cells/mL. The pre-culture was maintained under the same conditions as the test. Pre-culture medium: M2, according to OECD 201.
- 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 21 - 24 °C, with a light intensity of 60 - 120 µE/m2/s (400 - 700 nm) in M1 medium (according to NPR 6505, "Nederlandse Praktijk Richtlijn no. 6505)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/L (Ca+Mg) = 24 mg CaCO3/L (Pre-culture medium)
Test temperature:
21 - 23 °C
pH:
Control: 8.1 (0 h) and 8.4 (72 h)
Limit concentration 8.1 (0 h) and 8.3 (72 h)
Nominal and measured concentrations:
0.10, 1.0, and 10 mg/L (nominal, range-finding test)
Control and 100 mg/L (nominal, limit test)
Control and 83 -99 mg/L (measured, limit test)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass vessels with perforated aluminium caps filled with 50 mL test solution
- Initial cell density: 1.0E+04 cells/mL
- Control end cell density: 216.2E+04 cells/mL
- No. of vessels per concentration (replicates): 6 (limit concentration), 3 (range-finding concentrations)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, M1 according to NPR 6505 (stock culture) and M2 according to OECD 201 (pre-culture and test)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium was prepared with Milli-RO water (tap water purified by reverse osmosis, Millipore Corp, USA)
- pH: 8.1 ± 0.3
- Culture medium different from test medium: The pre-culture medium and test medium were the same (M2 according to OECD 201).
- Intervals of water quality measurement: pH was measured at the beginning (0 h)and at the end (72 h) of the test for the limit concentration and the control. The temperature of the medium was continuously mesured in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination with TLD lamps
- Light intensity and quality: 97 - 98 µE/m2/s

EFFECT PARAMETERS MEASURED:
- Cell density: At 24-h intervals (control and limit concentration), after 72 h (intermediate concentrations)
- Determination of cell concentrations: By microscope and counting chamber (0 h) and by spectrophotometry using an immersion probe with a path length of 10 mm
- Appearance of the cells: At the end of the test by microscope (control and limit concentration)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: Yes, combined limit/range-finding test
- Test concentrations: Control, 0.10, 1.0 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, at the end of the test, no significant growth rate or yield inhibition was recorded at any exposure concentration.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7, Merck)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: Yes
- Observation of abnormalities: No
- Any stimulation of growth found in any treatment: Very low (-0.43 and -0.54% inhibition in 0.10 and 10 mg/L treatment)
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the result was within the historical ranges for growth rate inhibition.
- ErC50 (72 h) = 1.6 mg/L (95% CI: 1.5 - 1.6 mg/L
- Other: The reference test was carried out 19 - 22 Mar 2018 according to OECD guideline 201 using potassium dichromate (K2Cr2O7) as reference substance.
Reported statistics and error estimates:
The NOEC was determined according to OECD guideline 201. An effect was considered to be significant if statistical analyses of the data obtained for the limit concentration revealed significant inhibition of growth compared to the negative control (two-sample t-Test, alpha = 0.05, one-sided, smaller).
Test for normality of distribution: Shapiro-Wilk's Test on Normal Distribution.
Test for homogeneity of variance: Levene's Test on Variance Homogeneita (with Residuals)
NOEC: Two-sample t-test Procedure

Any other information on results incl. tables

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline (Table 1) and is thus considered reliable and valid.

 

Table 1: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

In the control, cell density increased by an average factor of 216.

Yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

The mean coefficient of variation for section-by-section specific growth rates in the control was 21%.

Yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.0%.

Yes

 

ANALYTICAL RESULTS

The actual concentration measured in the limit concentration was 83 – 99% relative to nominal throughout the exposure period. The concentrations measured in solutions with algae were comparable to those measured in the solutions without algae (Table 2). Thus, the presence of algae did not affect test item concentrations in the test solutions. Based on these findings, the effect parameters were expressed in terms of the analytically confirmed nominal test item concentration.

 

Table 2. Analytical results.

Sampling time

Concentration [mg/L]

Relative to nominal

Relative to initial

[h]

nominal

analyzed

[%]

[%]

0

0

100

1001

n.d.

98.8

88.9

n.a.

99

89

 

24

0

100

1001

n.d.

87.8

91.5

n.a.

88

92

n.a.

89

103

72

0

100

1001

0.01172

82.7

83.3

n.a.

83

83

n.a.

84

94

1Without algae

2Maximum contribution to the samples at the concentration level of 100 mg/L was 0.014%.

n.d. Not detected

n.a. Not applicable                                       

 

BIOLOGICAL RESULTS

No inhibition of growth rate or yield was recorded at any of the tested concentrations (Table 3). Microscopic observations at the end of the test did not detect any aberrations in appearance up to the limit concentration of nominally 100 mg/L compared to the control. Effect parameters are summarized in Table 4.

 

Table 3. Growth Rate and Percentage Inhibition at Different Time Intervals.

Nominal concentration

 

n

 

0 – 24 h

 

24 – 48 h

 

48- 72 h

[mg/L]

mean

% inhibition

mean

% inhibition

mean

% inhibition

Control

6

2.212

 

1.700

 

1.464

 

100

6

2.279

-3.1

1.625

4.4

1.463

0.063

 

Table 4. Summary of obtained effect values (nominal).

ErC50 (72 h)

> 100 mg/L

EyC50 (72 h)

> 100 mg/L

NOEC (72 h)

for growth rate and yield based on statistical significance and on biological relevance

≥ 100 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.