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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:
From August 2017 to April 2018
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)
Deviations:
yes
Remarks:
without any impact on the overall integrity of the study or the interpretation of the study results and conclusions
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Appearance: Pink liquid
- Test item storage: At room temperature protected from light
- Purity/Composition correction factor: No correction factor required
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
- frequency: at t = 0h, t = 24h and t = 72h;
- volume: 0.80 mL;
- storage: samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
At the end of the exposure, the replicates were not pooled at each concentrations before sampling. Instead, samples were taken from the same replicate at each test concentration.

Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Vehicle:
no
Details on test solutions:
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the 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 the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Stock culture : Algae stock cultures were started by inoculating growth medium (M1 medium formulated using Milli-RO water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture (adjusted M2 medium formulated using Milli-RO water) were inoculated in culture medium at a cell density of 10000 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
180 mg/L (expressed as CaCO3)
Test temperature:
22 - 23°C
pH:
7.4 - 7.9
Nominal and measured concentrations:
Nominal concentrations (final test): solutions containing 0 (control), 1, 3.2, 10, 32 and 100% of the Saturated Solution prepared at a loading rate of 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL, all-glass, airtight closed vessels with no headspace
- Renewal rate of test solution: none (static system)
- Initial cells density: 1 x 10000 cells/mL
- Control end cells density: 175.1 x 10000 cells/mL
- No. of vessels per concentration (replicates): 3 (+ 1 extra replicate for sampling purposes after 24 hours of exposure + 1 or 2 replicates of each test concentration without algae)
- No. of vessels per control (replicates): 6
- Incubation: Closed screw cap septum vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking

TEST MEDIUM / WATER PARAMETERS
- Medium: Adjusted M2 medium

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: continous illumination
- Light intensity and quality: 85-87 μE.m-2.s-1

EFFECT PARAMETERS MEASURED
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions. Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period

OTHER PARAMETERS MEASURED
- pH: at the beginning and at the end of the test in the control and the highest test concentration
- Temperature of medium: continuously in a temperature control vessel
- Appearance of the cells: at the end of the test, microscopic observations were performed on the control and the limit concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS:
- Range finding test: yes (solutions containing 0 (control), 1, 10 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.39 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 0.38-0.40 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 0.13-0.14 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Remarks on result:
other: 95% CI 0.14-0.16 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.058 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Remarks on result:
other: 95% CI 0.047-0.067 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.054 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Remarks on result:
other: based on biological relevance
Results with reference substance (positive control):
The 72h-ErC50 for growth rate inhibition by potassium dichromate was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Calculation of ECx values was based on concentrations up to 32% of the SS because no TWA concentration could be calculated for the highest test concentration. Probit analysis was conducted using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item.

- Test for normality of distribution; Shapiro-Wilk´s Test on Normal Distribution
Test for homogeneity of variance: Levene´s Test on Variance Homogeneity
Determination of NOEC: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Measured Test Item Concentrations

At the start of the test, the actual test concentrations were 0.063, 0.13, 0.39 and 2.33 mg/L in solutions containing 1.0, 3.2, 10 and 32% of the SS (see below table). Unexpectedly, no detectable test item concentrations were found in the samples taken from the undiluted SS. This is most likely due to an error during the sample preparation or the instrumental analysis. At the end of the exposure period, the measured concentrations had decreased to 15-22% of the initial concentrations. The concentrations measured in the samples without algae were generally comparable to the corresponding results of samples with algae. Based on these results, the TWA concentrations were calculated to determine the effect parameters. No TWA concentration could be calculated for the highest test concentration.

Measured concentration (mg/L)

t=0h

Measured concentration (mg/L)

t=24h

Measured concentration (mg/L)

t=72 h

TWA (mg/L)

1.0

0.0634

0.0307

0.00922

0.026

3.2

0.129

0.065

0.0186

0.054

10

0.394

0.262

0.0865

0.21

10 without algae       

0.674

0.283

0.204

0.31

32

2.33

1.14

0.292

0.93

100

n.d.

4.62

0.520

n.a.

Inhibition of Growth Rate

Growth rates were in the range of the control for the lowest test concentration during the 72-hour test period, whereas growth rates of algae exposed to TWA concentrations of 0.054  

mg/L and higher were increasingly reduced (see below table). Statistically significant inhibition of growth rates was found at a TWA concentration of 0.054 mg/L and higher. However, the effects observed at TWA concentrations of 0.054 mg/L were considered not biologically relevant and, therefore, the NOEC based on biological relevance was set to 0.054 mg/L.

TWA

concentration

[mg/L]

Mean

Growth rate

[day-1]

Std. Dev.

[day-1]

n

%Inhibition

Control

1.722

0.0118

6

 

0.026

1.717

0.0139

3

0.3

0.054

1.693

0.0282

3

1.7#

0.21

1.336

0.0101

3

22.4*

0.93

0.253

0.0216

3

85.3*

n.d. (100% SS)

0.346

0.0399

3

79.9*

* - effect was statistically significant,

#- effect was statistically significant but biologically not relevant (<10%).

Inhibition of Yield

Inhibition of yield increased with increasing concentrations of the test item from a TWA concentration of 0.054 mg/L upwards, resulting in 99% inhibition at TWA concentrations of

0.93 mg/L and higher. Statistically significant inhibition of yield was found at TWA concentrations of 0.054 mg/L and higher. However, the effects observed at a TWA concentration of 0.054 mg/L were considered not biologically relevant and, therefore, the NOEC based on biological relevance was set to 0.054 mg/L

TWA

concentration

[mg/L]

Mean

Yield

[day-1]

Std. Dev.

[day-1]

n

%Inhibition

Control

174.1

6.14

6

 

0.026

171.9

7.12

3

1.3

0.054

159.9

13.76

3

8.1#

0.21

54.0

1.68

3

69.0*

0.93

1.1

0.14

3

99.3*

n.d. (100% SS)

1.8

0.35

3

98.9*

* - effect was statistically significant,

#- effect was statistically significant but biologically not relevant (<10%).

Acceptability of the Test

1. In the controls, cell density increased by an average factor of at least 16 within the exposure period (i.e. 175)

2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 11%)

3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 0.7%)

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of test conditions, a dose-related increase of inhibition of growth rates was observed from a TWA concentration of 0.054 mg/L upwards. The EC10 for growth rate inhibition (72h-ERC10) was 0.14 mg/L with a 95% confidence interval ranging from 0.13 to 0.14 mg/L. The EC50 for growth rate inhibition (72h-ERC50) was 0.39 mg/L with a 95% confidence interval ranging from 0.38 to 0.40 mg/L. The 72h-NOEC for growth rate inhibition was 0.054 mg/L based on biological relevance and 0.026 mg/L based on statistical significance.
Executive summary:

The toxicity of the test item to the freshwater green alga Pseudokirchneriella subcapitata was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201. Under the conditions of the test, a dose-related increase of inhibition of growth rates was observed from a TWA concentration of 0.054 mg/L upwards. The EC50 for growth rate inhibition (72h-ErC50) was 0.39 mg/L (95% confidence interval ranging from 0.38 to 0.40 mg/L). The EC10 for growth rate inhibition (72h-ErC10) was 0.14 mg/L (95% confidence interval ranging from 0.13 to 0.14 mg/L). The 72h-NOEC for growth rate inhibition was 0.054 mg/L based on biological relevance and 0.026 mg/L based on statistical significance.

Description of key information

The toxicity of the test item to the freshwater green alga Pseudokirchneriella subcapitata was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201. Under the conditions of the test, a dose-related increase of inhibition of growth rates was observed from a TWA concentration of 0.054 mg/L upwards. The EC50 for growth rate inhibition (72h-ErC50) was 0.39 mg/L (95% confidence interval ranging from 0.38 to 0.40 mg/L). The EC10 for growth rate inhibition (72h-ErC10) was 0.14 mg/L (95% confidence interval ranging from 0.13 to 0.14 mg/L). The 72h-NOEC for growth rate inhibition was 0.054 mg/L based on biological relevance and 0.026 mg/L based on statistical significance.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.39 mg/L
EC10 or NOEC for freshwater algae:
0.14 mg/L

Additional information

The toxicity of the test item to the freshwater green alga Pseudokirchneriella subcapitata was investigated in a GLP-compliant study performed in accordance with standard methods, with minor deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.