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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jun - 01 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted Mar 2006, corrected 28 Jul 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
other: SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of preregistration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany (24 Sep 2013)
Analytical monitoring:
yes
Remarks:
HPLC-UV/VIS
Details on sampling:
- Concentrations: Control, 2.6, 6.4, 16, 40, and 100 mg test item/L (nominal) at 0 h and 72 h
- Sampling method: Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at test start (0 h) and test end (72 h). At test end the contents of the test beakers were pooled. All samples were diluted by a factor of two with acetonitrilie.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C) protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 50.0 mg test item into 500 mL test water by ultrasonic treatment for 7 min and intense stirring for 2 h. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentrations.
- Controls: Test water (reconstituted water: OECD medium) without test item.
- Evidence of undissolved material: There were no remarkable observations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater green algae
- Strain: No. 61.81 SAG
- Age of inoculum (at test initiation): The cells for the test were taken from an exponentially growing pre-culture, which was set up 4 d prior to the test start under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in the testing facility under standardised conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L = 24 mg/L CaCO3 (calculated)
Test temperature:
22.5 - 23.0 °C
pH:
6.7 - 8.0 (0 h)
8.2 - 9.3 (72 h)
Nominal and measured concentrations:
Control, 2.6, 6.4, 16, 40, and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: Covered with glass dishes
- Material, size, headspace, fill volume: Size: 50 mL; Fill volume: 50 mL
- Initial cells density: 5000 cells/mL
- Control end cells density: 92.310 [10000 cells/mL]
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes (reconstituted water: OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The pH was measured in all test item concentrations and the control at the start and at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was adjusted with 1 M HCl to pH 8.0.
- Photoperiod: Continuous
- Light intensity and quality: 5623 lux (mean)
- Other: The flasks were incubated in a water bath, placed in a random order and were repositioned each day to minimize differences in test conditions.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Spectrophotometer (24, 48, 72 h)
- Microscopic examination (72 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.46 - 2.5
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
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, 184.6-fold increase within 72 h.
- Observation of abnormalities: No abnormalities observed.
- Any observations that might cause a difference between measured and nominal values: No remarkable observations, clear test medium.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50 (72 h) = 1.50, 95% confidence interval: 1.43 - 1.58 mg/L (nominal)
- Other: Reference test dating February 2016
Reported statistics and error estimates:
Based on the calculated cell densities the 72 h EyC50, EyC20, EyC10 and their 95%-confidence limits were calculated by Probit analysis. The 72 h ErC10 was calculated by Probit analysis. The ErC50 and ErC20 could not be calculated by Probit analysis due to the lack of effects and were therefore determined directly from the raw data. For the determination of the 72 h LOEC and the 72 h NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test.

ANALYTICAL RESULTS

At the start of the test 105% of the nominal test concentrations were found (average of all test concentrations). After 72 h test duration, 99% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 102% of nominal. Therefore, all reported results refere to nominal concentrations.

BIOLOGICAL RESULTS

The EyC50 (72 h) was > 100 mg test item/L. The calculated EyC10 (72 h) was 4.94 mg test item/L. The NOEyC (72 h) was 6.4 mg test item/L and the associated LOEyC (72 h) was 16 mg test item/L. The NOErC (72 h) was 6.4 mg test item/L and the associated LOErC (72 h) is 16 mg test item/L. Results are summarized in Table 1.

 Table 1: Growth Rates µ and Percentage Inhibition of µ during the Test Period.

Nominal concentration

[mg test item/L]

Growth rates µ [1/day] and % inhibition of µ

0 – 24 h

0 – 48 h

0 – 72 h

µ

%

 

µ

%

 

µ

%

 

Control

1.831

-

 

1.772

-

 

1.739

-

 

2.6

1.758

4.0

 

1.703

3.8

 

1.737

0.1

 

6.4

1.830

0.0

 

1.751

1.2

 

1.709

1.7

 

16

1.783

2.6

 

1.686

4.8

*

1.659

4.5

*

40

1.678

8.3

*

1.692

4.5

*

1.642

5.5

*

100

1.562

14.7

*

1.488

16.0

*

1.566

9.9

*

Negative values in `% inhibition´indicate an increase in growth relative to that of the control.

* mean value significantly different from the control (tested with Williams t-test, alpha = 0.05, one-sided)

The validity criteria were met:

Coefficient of variation of sectional (daily) growth rates in control cultures:  5.5 %.

Coefficient of variation of average growth between control replicates:  1.6 %.

Description of key information

ErC10 (72 h) > 100 mg/L (nominal, OECD 201, P. subcapitata)

ErC50 (72 h) > 100 mg/L (nominal, OECD 201, P. subcapitata)

Key value for chemical safety assessment

Additional information

There is one study available, in which the toxicity of N-Acetyl-DL-tryptophan (CAS 87-32-1) to aquatic algae was assessed according to OECD guideline 201 and GLP.

In a static test, Pseudokirchneriella subcapitata with an initial cell density of 5000 cells/mL was exposed to nominal test item concentrations of 2.6, 6.4, 16, 40, and 100 mg /L for 72 h. Defined volumes of the algal suspensions were sampled after 24, 48, and 72 h for the determination of cell densities by spectrophotometrical measurement. Test item concentrations were analytically verified by HPLC-UV/VIS at the start (0 h) and at the end of the test (72 h).

At the start of the test 105% (mean) of the nominal test concentrations were found. After 72 h test duration, 99% (mean) of the nominal value was determined, indicating that the test item was stable throughout the test. Therefore, all reported results refer to nominal concentrations. The calculated ErC10 (72 h) and ErC50 (72 h) were both > 100 mg test item/L (nominal).