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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:
20 Aug - 23 Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

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, Annex 5 corrected: 28 July 2011
Deviations:
yes
Remarks:
see detail information in "Any other information on results inclusive tables".
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
adopted 07. December 2015
Deviations:
yes
Remarks:
see detail information in "Any other information on results inclusive tables".
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Mainz, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test item concentration and the control were tested at test start and test end.
- Sampling method: Samples were diluted to the calibrated range with respective medium.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution containing 100.0 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments (0.32, 1, 3.2, 10 and 32 mg/L) were prepared by dilution of this stock solution with algal medium.
- Controls: Yes, medium without test item

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: SAG 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen, Germany), obtained in January 2016
- Age of inoculum (at test initiation): 96 h (pre-culture)
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Acclimation period: 96 h (pre-culture)
- Culturing media and conditions (same as test or not): For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 21.1 – 23.1 °C). The resulting culture grew exponentially.

Study design

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

Test conditions

Test temperature:
21.4 – 23.4 °C
pH:
7.1 - 7.8 (Control)
7.1 - 7.8 (Test item concentrations)
Nominal and measured concentrations:
Control, 0.32, 1, 3.2, 10, 32 and 100 mg/L (nominal concentration)
0, 0.3 and 1.0 (based on the nominal test item concentration since the measured conentration was below the LOQ), 3.1, 9.5, 30.5 and 95.4 mg/l (based on the geometric mean)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: Glass flasks, total volume 65 mL, each filled with 45 ± 1 mL
- Initial cells density: cell concentration of 2.3 *10E+03 cells/mL
- Control end cells density: 494,950 *10E+03 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: medium according to OECD guideline
- Culture medium different from test medium: medium of pre-culture same as for the test
- Intervals of water quality measurement: At test start and test end

OTHER TEST CONDITIONS
- Photoperiod: Continuously
- Light intensity and quality: 4440 – 8880 Lux

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study: yes (non-GLP)
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: not reported
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.61 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: Yes
- Observation of abnormalities: No cells were visible at 32 and 100 mg/L
Results with reference substance (positive control):
- Results with reference substance valid?
- EC50 (72 h) for growth rate: 0.66 mg/L (95% confidence interval: 0.64 - 0.69 mg/L)
- Other: The study (201901R301) was performed under GLP conditions in February 2019.
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.3.0. Since the Williams test could not find significance for growth rate, the Dunett´s Multiple ttest was set manually to calculate a NOEC.

Any other information on results incl. tables

Deviations from the Guideline:

The stock solutions to be autoclaved were autoclaved for 20 min instead of the required 15 min. This can be considered uncritical, as the goal of sterility is achieved.

The pre-culture was prepared 5 days before the test start. As all validity criteria were met and exponential growth was observed in the blank control, this was stated as uncritical.

The deviations were assessed and signed by the study director on 05. Sep. 2019.

Analytical results:

The measured concentrations lay between 89% and 112% of the nominal concentrations at the beginning of the test and between 62% and 110% of the nominal concentrations at the end of the test. Therefore, the determination of the results was based on the geometric mean of the measured concentrations (see § 39 OECD 201).

Because no test item could be measured or the measured concentration lay below the LOQ at the two lowest concentrations (0.32 / 1 mg/L nominal), no geometric mean could be calculated for these concentrations. Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the measured concentration in the highest concentrated treatment and the respective dilution factor. According to the guideline OECD 201 analysis of the results should be based on geometric mean concentration during exposure or on models describing the decline of the concentration of the test substance, if the deviation from the nominal or measured initial concentration is not within the range of ± 20 %.

Table 1: Cell numbers/mL

Nominal Concentration in [mg/L]

Parameter

Cell Number/mL

0 h

24 h

48 h

72 h

Blank control

Mean

2260

7413

62417

494950

SD

0

696

9987

13727

 

0.32

Mean

2260

5353

35960

439000

SD

0

771

53

27798

 

1

Mean

2260

6013

34007

434260

SD

0

110

6245

36877

 

3.2

Mean

2260

5860

43433

464140

SD

0

451

8932

5123

 

10

Mean

2260

4833

33093

208407

SD

0

151

2660

51226

 

32

Mean

2260

2320

2093

7287

SD

0

208

335

5451

 

100

Mean

2260

1913

1860

2100

SD

0

443

314

386

 

 

Table 2: Measured Concentrations and analytical raw data

Nominal Concentration Test Item

Area [mAU*min]

Measured conc. without accuracy in [mg/L]

Mean of measured

conc. with accuracy in [mg/L]

[mg/L]

t = 0 h

t = 72 h

t = 0 h

t = 72 h

t = 0 h

t = 72 h

5 (QC sample)

2.1215

2.1690

5.17

5.27

--

--

 

2.0284

2.1863

4.96

5.31

Blank control

n.a

n.a

--

--

--

--

 

n.a

n.a.

--

--

0.32

0.2957

0.2729

<LOQ

<LOQ

< LOQ

< LOQ

 

0.2578

0.2999

<LOQ

<LOQ

1

0.5211

0.2676

1.15

<LOQ

0.89

--

 

0.3979

n.a.

<LOQ

--

3.2

1.5503

1.6210

3.87

3.84

3.57

3.53

 

2.0803***

--

5.08***

--

 

1.5621

1.6214

3.90

3.84

10

4.7734

3.0645

10.12

6.75

9.31

6.20

 

4.7907

3.0584

10.15

6.74

 

 

32

3.5218*

4.6396*

31.81

37.65

29.29

34.63

 

3.5357*

4.6489

31.92

37.71

 

 

100

4.6412**

5.6809**

99.00

109.84

90.10

101.10

 

4.5244**

5.7021**

97.06

110.15

 

 

5 (QC sample)

1.9541

2.1538

4.80

5.24

 

 

 

1.9236

2.1420

4.73

5.21

--

--

LOQ (Limit of quantification) = 1 mg/L lowest calibration level

* including dilution factor 4

** including dilution factor 10

*** values were not used for the analytical determination, error in the measurement

n. a. = not analysable

Table 3: % of Nominal Concentration, Geometric mean and calculated Concentrations

Nominal Concentration Test Item

% of Nominal Concentration

*Geometric Mean

** Calculated Concentration

mg/L

t = 0 h

t = 72 h

mg/L

mg/L

Blank control

--

--

--

--

0.32

--

--

--

0.3

1

89

--

--

1.0

3.2

112

110

3.6

3.1

10

93

62

7.6

9.5

32

92

108

31.8

30.5

100

90

101

95.4

95.4

* Geometric mean is calculated with the measured concentrations at the beginning and at the end of the study.

** Calculated concentration on the basis of the geometric mean of the measured concentration in the highest concentrated treatment and the respective dilution factor.

Table 4: Inhibition Values

Nominal concentration in mg/L %

 Inhibition

72 h)

Growth Rate (0-

Yield (0-72 h)

Blank control

0

0

0.32

2.24

11.36

1

2.47

12.32

3.2

1.19

6.25

10

16.40

58.16

32

81.94

98.98

100

101.56

100.03

Inhibition higher than 100 % means a reduction of the algal cell concentration compared to the start of the test.

Table 5: Biological Results of the Test Item

Parameter

Value

 95% confidence interval

NOEC (Growth Rate) 72 h

3.1 mg/L

 --

NOEC (Yield) 72 h

1.0 mg/L

 --

LOEC (Growth Rate) 72 h

9.5 mg/L

--

LOEC (Yield) 72 h

3.1 mg/L

--

72h ErC10

8.12 mg/L

6.69 mg/L – 9.85 mg/L

72h EyC10

5.12 mg/L

1.93 mg/L – 13.59 mg/L

72h ErC50

17.61 mg/L

14.17 mg/L – 21.93 mg/L

72h EyC50

8.94 mg/L

2.68 mg/L – 28.66 mg/L

 

Table 6: Validity criteria

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.

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%

31%

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%.

1%

Yes

Applicant's summary and conclusion

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