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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:
23 - 26 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L (nominal) was prepared by heating the solution to 56 °C and stirring for 15 min until the test item was fully dissolved. The test concentrations were prepared by dilution of the stock solution with nutrient growth medium (aliquots of 1.6 - 160 mL stock solution were filled up to 500 mL with nutrient growth medium).
- Controls: Test medium without test item under same test conditions.
- Evidence of undissolved material: The stock solution was clear and colourless. Foam was observed during the preparation of the stock solution (during heating and stirring) but no foaming was recorded during pipetting or during the preparation of the dilutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater alga
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Scottish Marine Institute, Oban, Scotland, UK
- Method of cultivation: Algae were cultured in deionised water with added nutrients according to OECD 201 at 22.0 - 22.2 °C, 6440 - 7920 lux continuous white light and orbiting at 200 rpm.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.0 - 24.0 °C (test solution)
21.7 - 23.9 °C (incubator)
pH:
7.73 - 8.10
Nominal and measured concentrations:
Control, 0.32, 1.0, 3.2, 10.0, and 32.0 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical flask filled with 100 mL test volume
- Initial cell density (0 h): 1.0E04 cells/mL (mean)
- Control end cell density (72 h): 150.9E4 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, sterilised deionised water with added nutrients according to OECD guideline 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water for the preparation of the culture/test media was prepared by sterilization (autoclaving at 120 °C for 15 min) of de-ionised, filtered water.
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: The temperature and light intensity in the incubator were recorded at 0, 24, 48, and 72 h. The pH and temperature in the test solution were recorded at the beginning of the test (0 h) in each replicate and at the end of the test (72 h) in pooled replicates.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the control medium was adjusted from 7.80 to 7.10.
- Photoperiod: Continuous light
- Light intensity: 6440 - 7920 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Direct cell counts using a haemocytometer and microscope after 0, 24, 48, and 72 h.
- Other: Morphological abnormalities by microscopy after 0, 24, 48, and 72 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125 - 3.2
- Range finding study: Yes
- Test concentrations: 0.1, 1.0, 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes, after 72 h 94% inhibition was observed at the 100 and 1000 mg/L treatment levels and 75% at the 10 mg/L concentration level.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.448 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 2.00 -6.40mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13.47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 10.56 - 17.71 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 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 morphological abnormalities
- Any stimulation of growth found in any treatment: Stimulation of growth rate was observed at the 0.32 and 1.0 mg/L treatment levels after 72 h.
- Any observations that might cause a difference between measured and nominal values: All test concentrations appeared clear and colourless.
- 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): 1.188 mg/L (potassium dichromate) with 95% confidence limits of 0.979 - 1.444 mg/L
- Other: Exposure from 10 - 13 May 2016
Reported statistics and error estimates:
EC50 values were estimated graphically where possible and 95% confidence limits were calculated according to the method of ToxCalc Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software (copyright 1994 - 1996).
The EC50 for growth rate and yield inhibition were determined by Maximum Likelihood-Probit/Logit, respectively.

VALIDITY CRITERIA

The study fulfilled the validity criteria laid down by the OECD guideline 201 (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.

The control cell density increased by a factor of 150.9 in 72 h (specific growth rate of 1.66/d).

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 variation coefficient for the section-by-section specific growth rates in the control cultures was 23.37%.

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.

The variation coefficient for the 72 h period of the control cultures was 4.59%.

Yes

 

ANALYTICAL RESULTS

No analytical confirmation of the test concentrations was performed. The dissolved concentrations were likely to correspond to the reported nominal concentrations. 

BIOLOGICAL RESULTS

Results are summarized in Tables 2 and 3. The LOEC (72 h) was 10 mg/L (Bonferroni t-Test).

Table 2. Percent inhibition.

Nominalconcentration(mg/l)

Percent inhibition by yield

Percent inhibition by growth rate

0 - 48 hours

0 - 72 hours

0 - 48 hours

0 - 72 hours

0.32

15

-2

5

-1

1.0

-26

-9

-7

-1

3.2

11

16

3

3

10.0

85

84

53

37

32.0

100

99

100

84

 

Table 3. Effect values by yield (EyCx) and growth rate (ErCx)

Exposure Period (hours)

EyCxvalue mg/l (95% confidence limits)

ErCxvalue mg/l (95% confidence limits)

EyC10

EyC20

EyC50

ErC10

ErC20

ErC50

0 to 48

3.139

(2.329-3.822)

3.991

(3.161-4.700)

6.016

(5.170-6.899)

 

4.825 *

 

6.101*

 

9.556*

0 to 72

2.663

(1.906-3.297)

3.510

(2.729-4.186)

5.627

(4.781-6.631)

4.448

(2.000-6.403)

6.695

(3.907-8.818)

13.47

(10.56-17.71)

NOEC

(0-72h)

3.2mg/l

(Determined by Wilcoxon Two-Sample Test$)

3.2mg/l

(Determined by Bonferroni t Test$)

* Confidence limits not possible to determine.

All concentrations of the test substance are reported as nominal as received.

Methods used in Toxcalc v5.0: 48 hour ErCxdetermined by Maximum Likelihood-Probit and 48 hour EyCxdetermined by Maximum Likelihood-Logit. 72 hour ErCxand EyCxboth determined by Maximum Likelihood-Logit.

$Following Shapiro-Wilk’s Test for normal distribution and Bartlett’s Test for equal variances. Equality of variance could not be confirmed for 72 hour yield.

 

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

Description of key information

ErC50 (72 h) = 13.47 mg/L (nominal, OECD 201, P. subcapitata)

ErC10 (72 h) = 4.448 mg/L (nominal, OECD 201, P. subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:
13.47 mg/L
EC10 or NOEC for freshwater algae:
4.448 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Pseudokirchneriella subcapitata. The first main test did not meet the validity criteria as the coefficient of variation of the replicates for the control was > 7% for the 72-hour test period. Therefore, this main test was repeated. All validity criteria for the second main test were met. The algae cells were exposed for a period of 72 h to nominal test concentrations of 0.32, 1.0, 3.2, 10.0, and 32.0 mg/L. No analytical measurement was conducted. After72 h inhibition of growth was observed in the two highest test concentrations (10 and 32 mg/L) resulting in an ErC50 (72 h) of 13.47 mg/L (nominal) and a ErC10 (72 h) of 4.448 mg/L (nominal).