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:
03 - 06 Dec 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Considerable deficiencies in test medium preparation and analytical method.

Data source

Referenceopen allclose all

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted 23 Mar 2006
Corrected 28 Jul 2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Adopted 07 Dec 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances And Mixtures
Version / remarks:
Adopted 14 Dec 2000
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Rheinland Pfalz, Germany (15.05.2018)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Remarks:
LC-MS-MS
Details on sampling:
- Concentrations: All concentrations, at test start (0 h) and every 24 h
- Sampling method: At test start, samples for the analytical determination were taken before the addition of the algal inoculum. Then, samples were taken every 24 h until test end (72 h). Algal cells were eliminated from the samples by centrifugation before analytical determination.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A water-accommodated fraction (WAF) was prepared by weighing the test item with the help of a cover glass into a corresponding amount of algal medium (demineralised water enriched with minerals) to obtain a stock WAF with a nominal concentration of 100.2 mg/L, which was then shaken at 100 rpm for 3 d on an orbital shaker. The test item was applied in thin layer to achieve a large surface for contact with aqueous medium. The resulting solution was centrifuged because the solution was turbid (deviation from study plan). The lower test item concentrations were prepared by dilution of this stock WAF with algal medium. The WAFs were prepared individually and not by serial dilution of the stock WAF (see "Any other information on materials and methods incl. tables").
- Differential loading: No
- Controls: Algal medium without test item

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: 86.81 (SAG number)
- Source: MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of University of Göttingen, Germany), Jan 2016
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From this stock culture, a permanent culture was prepared. The pre-culture for this test was prepared from an aliquot of the permanent culture.
- Age of inoculum at test start: 4 d.

Study design

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

Test conditions

Test temperature:
21.3 - 22.8 °C
pH:
Control: 7.6 (0 h) and 8.4 (72 h)
Treatments: 7.1 - 7.6 (0 h) and 7.6 - 8.1 (72 h)
Nominal and measured concentrations:
Control, 1.0, 3.2, 10, 32 and 100 mg/L (nominal loading rate WAF)
Control, 0.002, 0.006, 0.018, 0.058 and 0.183 µg/L (concentratins calculated based on geometric mean of the highest treatment)
Comment: After 24 and 48 h, the test item was only detectable in the highest concentration and at the end of the test, no test item was detected in any treatment. The measured concentration in the highest concentration at 48 h was slightly below the LOQ but was still used for the calculation of the geometric mean due to the clarity of its peak. The lower concentrations were derived from the geometric mean of the measured concentration in the highest treatment and the respective dilution factors. For details, refer to "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: 65 mL glass flasks were filled with 45 ± 1 mL test medium and covered with perforated plastic foil.
- Initial cell density: 2620
- Control end cell density: Mean = 339203, S.D. = 24513
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, the algal medium used corresponded to the OECD TG 201 medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algal medium was prepared with deionised water.
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: 0 and 72 h

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity: 5000 lux (0 - 72 h)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Electronic particle counter
- Measurement interval: 0, 24, 48 and 72 h
- Microscopical observations: The appearance of the cells was visually assessed at the end of the test (72 h).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes, non-GLP.
- Results used to determine the conditions for the definitive study: Yes, the test concentrations of the definitive test were based on the results of the non-GLP pre-test. Slight but statistically significant inhibition was observed at the loading rate of 1 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2 Cr2 O7, CAS 7778-50-9)

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Water Accommodated Fraction
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: Yes
- Observation of abnormalities: Normal and healthy appearance in all treatments. No cells were observed at the highest loading rate (100 mg/L).
- 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) for K2Cr2O7: 0.79 mg/L, 95% confidence interval: 0.79 - 0.79 mg/L
- Other: The EC50 (72 h) for potassium dichromate was determined in a separate reference test performed in January 2018 (GLP).
Reported statistics and error estimates:
Statistical analyses were conducted with ToxRat® Professional, v.3.2.1.
The following tests were applied: Shapiro-Wilk's Test on Normal Distribution, Levene's Test on Variance Homogeneity (with residuals), Trend analysis by Contrasts (Monotonicity of Concentration/Response), Williams Multiple Sequential t-test Procedure, Analysis of Variance and Test for Lack of Fit for the 3-param. normal CDF

Any other information on results incl. tables

VALIDITY CRITERIA

The study fulfilled the validity criteria laid down by the guideline (Table 1).

 

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.

Observed factor: 129

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%

Observed value: 30%

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. For other less frequently tested species, the value should not exceed 10%.

Observed value: 2%

Yes

 

ANALYTICAL RESULTS

At the start and after every 24 h, the test item concentrations were measured in the test media by LC-MS. However, in the present study a different LC-MS was used for the actual measurement than was used for the validation due to equipment failure.

 

MEASURED CONCENTRATIONS

At the beginning of the test (0 h), the test item could be analytically determined in samples from the 10 – 100 mg/L (nominal) treatments. Due to the very poor solubility, the test item was not detected in lower concentrations. After 24 and 48 h, the test item was only detectable in the highest concentration. At the end of the test, no test item was detectable in any treatment (Table 2).

 

CALCULATION OF THE GEOMETRIC MEAN MEASURED CONCENTRATIONS

The geometric mean was calculated by multiplication of the n concentrations and taking the nthroot thereof. According to the report, the measured concentration in the highest treatment at 48 h (0.15 µg/L) was slightly below the LOQ (0.2 µg/L) but because the measured peak of the test item was clear, it was still used for the calculation of the geometric mean of the highest concentration (Table 3). For the calculation of the geometric mean value of the highest test concentration, the test facility used half the LOQ (= 0.1 µg/L) for the 72 h – value. The geometric mean measured values for the lower concentrations were then derived from this calculated geometric mean of the highest test concentration (nominal 100 mg/L) using the respective dilution factors. The test facility justified this procedure with the preparation method of the lower concentration treatments, which were prepared by dilution of the stock WAF (100 mg/L nominal).

 

Due to considerable deficiencies in the preparation of test media and in the analytical method, only the results for the highest test concentration were considered in the present dossier.

 

 Table 2. Measured test item concentrations.

Nominal test item concentration

[mg/L]

Measured test item concentrations [µg/L]

0 h

24 h

48 h

72 h

Blank control

< LOQ

< LOQ

< LOQ

< LOQ

1.0

< LOQ

< LOQ

< LOQ

< LOQ

3.2

< LOQ

< LOQ

< LOQ

< LOQ

10

0.38

< LOQ

< LOQ

< LOQ

32

0.23

< LOQ

< LOQ

< LOQ

100

0.42

0.18*

0.15*

< LOQ

LOQ = 0.2 µL

* Value slightly below the LOQ but nonetheless used for evaluation due to the clarity of the measured peak.

 

Table 3. Geometric mean and calculated concentrations.

Nominal test item concentration

Geometric mean

Calculated concentration based on geometric mean

[mg/L]

[µg/L]

[µg/L]

Blank control

--

--

1.0

--

0.002

3.2

--

0.006

10

--

0.018

32

--

0.058

100

0.183

0.183

 

BIOLOGICAL RESULTS

Significant inhibition of algal growth was observed at 32 – 100 mg/L (nominal) (Table 4). Since the test item is not soluble, the EC50 value for growth rate inhibition is reported as a range instead of the exact value extrapolated by the software (Table 5 and 6).

Due to considerable deficiencies in the preparation of test media and in the analytical method, only the nominal EC50 (72 h) was considered as effect value in the present dossier.

 

Table 4. Inhibition Values at the end of the test (72 h)

Nominal test item concentration [mg/L]

Replicate

Inhibition of Growth Rate µ (72 h)

Inhibition of Yield (72 h)

Blank control

1

-0.65

-3.01

2

-1.47

-7.25

3

-1.49

-7.33

4

1.86

8.93

5

0.01

0.27

6

1.74

8.37

Mean

0

0

SD

1.50

7.28

1.0

1

3.24

14.88

2

-2.36

-11.99

3

2.68

12.53

Mean

1.19

5.14

SD

3.08

14.88

3.2

1

-2.27

-11.50

2

1.11

5.50

3

-2.02

-10.17

Mean

-1.06

-5.39

SD

1.88

9.45

10

1

3.95

17.80

2

0.43

2.30

3

-0.66

-3.05

Mean

1.24

5.68

SD

2.41

10.83

32

1

1.18

5.84

2

4.63

20.47

3

6.08

25.96

Mean

3.96

17.43

SD

2.52

10.40

100

1

43.36

88.56

2

47.63

90.85

3

52.66

93.01

Mean

47.88

90.81

SD

4.66

2.22

SD = standard deviation

 

Table 5. Summary of effect values (based on nominal values).

Parameter

Value

95% confidence interval

NOEC (72 h) – growth rate

10 mg/L

--

NOEC (72 h) – yield

10 mg/L

--

LOEC (72 h) – growth rate

32 mg/L

--

LOEC (72 h) – yield

32 mg/L

--

ErC10 (72 h)

44.57 mg/L

36.57 – 54.30 mg/L

EyC10 (72 h)

27.41 mg/L

20.48 – 36.67 mg/L

ErC50 (72 h)

> 100 mg/L

--

EyC50 (72 h)

51.85 mg/L

36.94 – 72.22 mg/L

 

Table 6. Summary of effect values (based on measured values).

Parameter

Value

95% confidence interval

NOEC (72 h) – growth rate

0.018 µg/L

--

NOEC (72 h) – yield

0.018 µg/L

--

LOEC (72 h) – growth rate

0.058 µg/L

--

LOEC (72 h) – yield

0.058 µg/L

--

ErC10 (72 h)

0.08 µg/L

0.07 – 0.10 µg/L

EyC10 (72 h)

0.05 µg/L

0.03 – 0.06 µg/L

ErC50 (72 h)

> 0.183 µg/L

--

EyC50 (72 h)

0.09 µg/L

0.08 – 0.12 µg/L

 

Applicant's summary and conclusion

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