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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:
10 Aug - 19 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
23 Mar 2006
Annex 5 corrected 28 Jul 2011
Qualifier:
according to
Guideline:
other: OECD series 23, Guidance document on aquatic toxicity testing of difficult substances and mixtures
Version / remarks:
2000
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Remarks:
UPLC
Details on sampling:
- Concentrations: A test concentrations and the control at 0, 24, and 72 h
- Sampling method: 1.2 mL from the approximate centre of the test vessels. Reserve samples of 1.2 mL were additionally taken from all test solutions.
- Sample storage conditions before analysis: In a freezer (≤ -15 °C) for a maximum of 3 months.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The preparation of test solutions started with a concentration of 100 mg/L and a 3-d period of magnetic stirring. The obtained mixture was then allowed to settle for a period of 3 h. Then, tha aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of this SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Test solution: After preparation, 50 mL were added to each replicate of the respective test concentration and 1 mL algal suspension was added at a final cell density of 1E+04 cells/mL.
- Differential loading: No
- Controls: Test medium without test item
- Evidence of undissolved material: The test item was a colourless to light yellow liquid and not completely soluble in test medium at the concentration initially prepared. No correction was made for the purity/composition of the test item. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
other: Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 M2 pre-culture medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as the test.
- 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 and a light intensity of 60 to 120 µE/m²/s (400 - 700 nm).

Study design

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

Test conditions

Hardness:
0.24 mmol/L (Ca+Mg) = 24 mg CaCO3/L (test medium)
Test temperature:
22 °C
pH:
8.2 - 8.3 (0 h)
8.1 - 8.4 (72 h)
Nominal and measured concentrations:
Control, 1.0, 3.2, 10, 32 and 100% of the saturated solution prepared at a loading rate of 100 mg/L
Control, 0.95, 3.1, 9.5, 32 and 97 mg/L (measured at t = 0 and 72 h)
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass test vessels with perforated aluminium caps filled with 50 mL test solution
- Initial cell density: 1.0 E+04 cells/mL
- Control end cell density: 230.70 E+04 cells/mL (mean of six replicates)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: The test vessels were randomly distributed in the incubator and repositioned daily. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: Yes, M1 (according to NPR 6505 "Netherlandse Praktijk Richtlijn no. 6505") and and M2 (according to OECD guideline 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 medium (test medium) was prepared according to OECD guideline 201 using Milli-RO water (tap water purified by reverse osmosis).
- Culture medium different from test medium: Culture medium same as test medium (M2).
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination using TLD-lamps
- Light intensity and quality: 85 - 86 µE*m^-2*s^-1

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: 24 h intervals by spectrophotometry (680 nm, immersion probe with a path length of 10 mm)
- Appearance of cells: At the end of the test (72 h) by microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx. 3.2
- Range finding study: Yes, a combined limit/range-finding test was performed before the final test
- Test concentrations: 1.0, 3.2, 10, 32 and 100% of the saturated solution prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, a concentration-related increase of algal growth inhibition was observed at all concentrations tested, resulting in 99 and 100% inhibition of growth rate and yield, respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 25 - 30 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
64 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 61 - 68 mg/L
Details on results:
- Exponential growth in the control: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the obtained effect concentration lies within the historical ranges for growth rate inhibition.
- ErC50 (72 h) = 0.90 mg/L with a 95% confidence interval from 0.88 - 0.93 mg/L
- Other: The reference test was conducted from 02 - 05 Jul 2018 according to OECD guideline 201.
Reported statistics and error estimates:
Statistical analyses were performed with ToxRat Professional v3.2.1 (ToxRat Solutions GmbH, Germany). The NOEC and ECx values were determined according to the OECD guideline 201 recommendations. Treatment vs control data were statistically compared by the one-side (smaller) Step-down Jonckheere-Terpstra Test procedure (growth rate) or the one-sided (smaller) Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (yield inhibition). The calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test item.

Any other information on results incl. tables

VALIDITY CRITERIA

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

 

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 231 within the exposure period.

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 cultures was 19%.

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 0.41%.

Yes

 

ANALYTICAL RESULTS

The measured test item concentrations at the start of the test were 0.95, 3.1, 9.5, 32 and 97 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the saturated solution prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 86 – 99% relative to the initial concentrations at the end of the test. The concentrations measured in the samples without algae were comparable to the concentrations measured in the samples with algae (Table 2).

A small concentration was measured in the control at the end of the test. It was concluded that contamination may have occurred during sampling. The measured value was below the measured value of the lowest calibration standard, with a contribution of 2.0% to the lowest test concentration. Consequently, the response was considered to be negligible and without consequences for the integrity of the present study.

 

Since all samples taken during the test were at 81 – 101% relative to the nominal concentrations, it was concluded that the undissolved material removed during the preparation of the test samples was not test item related. Thus, the effect concentrations were based on the analytically confirmed nominal concentrations.

 

Table 2. Analytical results.

Time of sampling

 

Percentage of saturated solution*

Analyzed concentration

Relative to initial

[h]

[%]

[mg/L]

[%]

 

0

0

n.d.

 

1.0

0.948

 

3.2

3.12

 

10

9.47

 

101

9.63

 

32

31.5

 

100

97.4

 

 

24

0

n.d.

n.a.

1.0

0.958

101

3.2

3.19

102

10

9.62

102

101

9.78

102

32

31.5

100

100

101

103

 

72

0

2

n.a.

1.0

0.811

86

3.2

2.78

89

10

9.01

95

101

8.83

92

32

30.6

97

100

96.5

99

*           Saturated solution prepared at a loading rate of 100 mg/L.

1            Without algae

2           Estimated value of 0.018 mg/L, calculated by extrapolation of the calibration curve (lowest calibration point 0.1 mg/L). Contamination might have occurred during sampling. The maximum contribution to the lowest test concentration, based on measured value and dilution factor, was 2.0%.

n.d.       Not detected

n.a.       Not applicable

 

 

BIOLOGICAL RESULTS

Inhibition of growth rates increased with increasing test item concentrations from 1.0 mg/L upwards, resulting in 75% inhibition at 100 mg/L. Statistically significant inhibition of growth rates was found at all test concentrations (Table 3 and Table 4). However, growth rate inhibition was not considered biologically relevant at 10 mg/L and lower, where the observed inhibition was < 10%. Thus, the NOEC based on biological relevance was set at 10 mg/L.

Inhibition of yield increased with increasing test item concentrations from 1.0 mg/L upwards, resulting in 99% inhibition at 100 mg/L (Table 5). Statistically significant inhibition of yield was found at all test concentrations.

The obtained effect values are summarized in Table 6.

 

Table 3. Growth rate inhibition.

Nominal test item concentration [mg/L]

 

Mean

 

S.D.

 

n

 

% inhibition

Control

1.814

0.0073

6

 

1.0

1.775

0.0066

3

2.1#

3.2

1.761

0.0065

3

2.9#

10

1.697

0.0106

3

6.4#

32

1.551

0.0453

3

14*

100

0.459

0.0986

3

75*

* Effect was statistically significant.

# Effect was statistically significant but not biologically relevant (< 10%)

 

Table 4. Growth rate inhibition at different time intervals.

Nominal test item concentration [mg/L]

 

n

 

0 – 24 h

 

24 – 48 h

 

48 – 72 h

mean

% inhibition

mean

% inhibition

mean

% inhibition

Control

6

2.183

 

1.771

 

1.486

 

1.0

3

2.237

-2.4

1.603

9.5

1.487

-0.057

3.2

3

2.142

1.9

1.672

5.6

1.470

1.1

10

3

2.022

7.4

1.613

8.9

1.455

2.1

32

3

1.890

13

1.396

21

1.368

8.0

100

3

1.439

34

0.011

99

-0.073

105

 

 Table 5. Yield inhibition.

Nominal test item concentration [mg/L]

 

Mean

 

S.D.

 

n

 

% inhibition

Control

229.7

5.07

6

 

1.0

204.7

4.08

3

11*

3.2

196.2

3.88

3

15*

10

161.4

5.20

3

30*

32

104.6

14.78

3

54*

100

3.1

1.23

3

99*

* Effect was statistically significant.

 

Table 6. Summary of effect concentrations (nominal concentrations, analytically confirmed)

Parameter [mg/L]

NOEC*

NOEC#

EC10

EC20

EC50

Growth rate

Value

< 1.0

10

27

37

64

 

lower 95%-cl

 

 

25

34

61

 

upper 95%-cl

 

 

30

39

68

Yield

Value

< 1.0

< 1.0

3.3

6.1

20

 

lower 95%-cl

 

 

2.8

5.5

19

 

upper 95%-cl

 

 

3.8

6.8

22

cI           Confidence limit

*            Based on statistical significance.

#            Based on biological relevance.

Applicant's summary and conclusion

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