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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:
15 july - 28 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006, Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Samples for possible analysis were taken from all groups.
- Frequency:at t=0 h, t=24 h and t=72 h.
- Volume:10 mL from the approximate centre of the test vessels.
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a WSF prepared at a loading rate of 100 mg/L 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 AND APPLICATION OF TEST SOLUTION

- Method: In the limit test, a WSF was prepared at a loading of 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of two hours. Thereafter, the aqueous WSF was collected by means of siphoning and used as the limit concentration.
- Evidence of undissolved material: The WSF was checked for Tyndall effects using a laser pen. No distraction of laser beam was observed indicating absence of undissolved material. All test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x E4 cells/mL
- 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.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23 and 24°C
pH:
8.2 - 8.4
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: WSF prepared at a loading rate of 100 mg/L
Measured concentrations: The concentration measured in the WSF prepared at 100 mg/L was 66 µg/L at the start of the test. During the first 24 hours of exposure, the initial concentration decreased to 0.81% relative to the initial concentration (0.53 µg/L) and was not detectable at the end of the test.Based on these results, the TWA concentrations was calculated to be 2.1 ug/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: No
- Initial cells density: An initial cell density of 1 x 104 cells/mL
- Control end cells density: A mean cell density in the controle of 286 x 104 cells/mL
- No. of vessels per concentration (replicates):6
- No. of vessels per control (replicates):6

GROWTH MEDIUM
- Culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:M2; according to the OECD 201 Guideline, formulated using Milli-RO water
- Culture medium different from test medium:yes
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH:no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 84 to 86 µE.m-2.s-1

EFFECT PARAMETERS MEASURED .
- Effecct parameters: 72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50, 72 h ErC10, 72 h EyC10
- Determination of cell concentrations: 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). Test medium was used as blank.

TEST CONCENTRATIONS

Range finding study:
- Test concentrations: exponentially growing algal cultures were exposed to WSFs prepared at 1.0, 10 and 100 mg/L and to a control.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7,)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.1 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF prepared at a lading rate of 100 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 2.1 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF preprared at a loading rate of 100 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.1 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF prepared at a loading rate of 100 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.
-Growth rate: Growth rate was inhibited by 2.4% at the end of the test in the limit concentration. This inhibition was found to be statistically significant due to low variability observed in the control treatment (0.27%). However, as this effect was lower than 10%, it was considered biologically not relevant. The NOEC based on biological relevance was thus 2.1 µg/L.
- Yield: An inhibition of 13% of yield was observed at the end of the exposure period in the limit concentrations. This effect was both statistically significant a biologically relevant. Therefore, neither a NOEC nor the EC10 can be given based on the results obtained in this study
Results with reference substance (positive control):
- Results with reference substance valid? yes
The 72h-EC50 for growth rate inhibition (ERC50) was 1.97 mg/L with a 95% confidence interval ranging from 1.93 to 2.00 mg/L.
Reported statistics and error estimates:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.
The ECx-values for growth rate inhibition could not be determined because the observed effects were below 10%. Since a limit test was performed, no ECx-values could be derived for yield inhibition.
For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test, α=0.05, one-sided, smaller).

Table 1          
Time Weighted Average Versus the Nominal Loading Rate

SHR 1396
Loading rate (mg/L)

Measured concentration (µg/L)

TWA conc. (µg/L)

t=0h

t=24h

t=72 h

100

66

0.53

0.0901

2.1

100 WA

68

0.26

0.084

1.5

1Half of the Limit of Detection (LOD = 0.18 µg/L), WA: without algae

Table 2          
Growth Rate and Percentage Inhibition for the Total Test Period

SHR 1396
TWA conc. (µg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.885

0.0051

6

 

2.1

1.839*

0.0052

6

2.4*

* effect was statistically significant however biologically not relevant (<10%).

Table 3          
Growth Rate and Percentage Inhibition at Different Time Intervals

 

SHR 1396
TWA conc. (µg/L) 

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

2.164

 

1.878

 

1.613

 

2.1

6

2.110

2.5

1.748

7.0

1.660

-2.9

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no biologically relevant inhibition of growth rate was recorded at the limit concentrations of SHR 1396 (i.e. 2.1 µg/L).
The 72h-EC50 for growth rate (ERC50) and yield inhibition (EYC50) was beyond the range tested, i.e. exceeded the TWA concentration of 2.1 µg/L.
The 72h-NOEC for growth rate inhibition was <2.1 µg/L based on statistical significance and 2.1 µg/L based on biological relevance.
The 72h-NOEC for yield inhibition was <2.1 µg/L based on both, statistical significance and biological relevance.
Executive summary:

A 72 hour toxicity study with freshwater algae (Pseudokirchneriella subcapitata) was conducted according to OECD guideline 201 and GLP prnciples.

The Algae were exposed to a Water Soluable Fraction (WSF) prepared at loading rate of 100 mg/L and a blank control (both 6 replicates). The concentration measured in the WSF prepared at 100 mg/L was 66 µg/L at the start of the test. During the first 24 hours of exposure, the initial concentration decreased to 0.81% relative to the initial concentration (0.53 µg/L) and was not detectable at the end of the test. The calculated Time Weight Average (TWA) concentration was 2.1 µg/L.

Growth rate was inhibited by 2.4% at the end of the test. This inhibition was found to be statistically significant, due to low variability observed in the control treatment (0.27%). However, as this effect was lower than 10%, it was considered biologically not relevant. The 72h-ErC50 and 72h-ErC10 were beyond the range tested.

The study met the acceptability criteria, and is considered to be valid.

Description of key information

There were no effects observed (NOEC) at the Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L ( and thus considered the maximum solubility of the testsubstance in medium) (TWA 2.1 µg/L). The EC10 was above the WAF prepared at 100 mg/L ( > TWA 2.1 µg/L).

Key value for chemical safety assessment

Additional information

A 72 hour toxicity study with freshwater algae (Pseudokirchneriella subcapitata) was conducted according to OECD guideline 201 an d GLP prnciples.

The algae were exposed to a Water Accommodated Fraction (WAF) prepared at loading rates of 100 mg/L and a blank control (both 6 replicates). The concentration measured in the WSF prepared at 100 mg/L was 66 µg/L at the start of the test. During the first 24 hours of exposure, the initial concentration decreased to 0.81% relative to the initial concentration (0.53 µg/L) and was not detectable at the end of the test. The calculated Time Weight Average (TWA) concentration was 2.1 µg/L.

Growth rate was inhibited by 2.4% at the end of the test. This inhibition was found to be statistically significant, due to low variability observed in the control treatment (0.27%). However, as this effect was lower than 10%, it was considered biologically not relevant. The 72h-ErC50 and 72h-ErC10 were beyond the range tested.

The study met the acceptability criteria, and is considered to be valid.