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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-Mar-2019 - 26-Aug-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 test concentrations and the control.
Frequency: at t=0 h, t=24 h and t=72 h.
Volume: 4.8 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.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 1.0 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.
Additionally, reserve samples of 4.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Preparation of test solutions started with the highest concentration of 100 mg/L in the range finding test and 10 mg/L in the final test, in low light conditions. No other treatment than vigorous shaking was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. Lower test solutions were clear and colorless at the end of the preparation procedure. From 1.0 mg/L upwards, solutions were clear and very light yellow to yellow except in the 1.0 mg/L in the range-finding test where the solution was colorless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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: 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 10^4 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:
no
Total exposure duration:
72 h
Hardness:
M2 medium: 0.24 mmol/L (24 mg CaCO3/L).
Test temperature:
between 22 and 23°C.
pH:
8.2 - 8.5
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
Measured concentrations:
- Measuremens: The measured concentrations at the start of the test were at 107 – 111% of nominal, except for the lowest test concentration which was below the limit of detection (LOD).During the exposure period, the concentrations decreased to 38 – 88% of initial at the end of the test. Time weight average concentrations were calculated.
-Concentrations (TWA): 0.042, 0.26, 0.78, 3.0, 9.6 mg/L.
It should be noted that a background response of the element was detected in the blank samples. The maximum contribution to the samples at the concentration level of 0.10 mg/L was 75% based on
response.
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 10^4 cells/mL.
- Control end cells density: A mean cell density in the controle of 111 x 10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- 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.
- Justification for the use of M2 medium: Potassium Ferricyanide contains Fe which is also present in M2 medium.For this reason, it is difficult to measure low test item concentrations in M2 medium. Therefore, the impact of the absence of FeCl3.6H2O in M2 medium on algal growth was evaluated. In conclusion, M2 medium without Fe did not provide sufficient nutrition to algae to allow them to reach the validity criteria prescribed by the guideline. Therefore, this study was continued with M2 medium with FeCl3.6H2O.
- Culture medium different from test medium: yes


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 88 to 89 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED .
- Effecct parameters: 72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50, 72 h ErC20, 72 h EyC20, 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. After 24 hours of exposure in the final test, and 48 hours of exposure in the range-finding test, undissolved particles were observed in the two highest test concentrations that disturbed spectrophotometric measurement. Therefore, algal density was determined by use of a microscope and a counting chamber during the tests.

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: exponentially growing algal cultures were exposed to concentrations of 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)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.042 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.67 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.50 - 0.90 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.4 - 2.0 mg/L
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 1.0 mg/L when compared to the control.
- Colour differences: at the end of the test, replicate 3 of 1.0 mg/L was visually lighter and less algae were observed in replicate 3 compared to the other two replicates. There was no significant difference in concentration between vessel 3 and pooled vessels 1 and 2.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: particles were observed in the two highest test item concentrations in the final test from 24 hours of exposure onwards. Those particles were observed microscopically and most likely test item related.
Results with reference substance (positive control):
Results with reference substance valid? : Yes
- EC50: 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:
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 negative control revealed significant inhibition of growth rate or inhibition of yield (Step-down Jonckheere-Terpstra Test, α=0.05, one-sided, smaller).
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 average exposure concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1          
Time Weighted Average Versus Nominal Concentrations

Potassium Ferricyanide

Nominal conc. (mg/L)

Measured concentration (mg/L)

TWA (mg/L)

t=0h

t=24h

t=72h

0.10

0.042*

0.042*

0.042*

0.042

0.32

0.34

0.32

0.16

0.26

1.0

1.1

0.94

0.48#

0.78

1.0WA

1.1

0.82

0.61

0.79

3.2

3.5

3.0

2.6

3.0

10

11

9.1

9.6

9.6

WA – Without Algae, * – Concentration was below the detection limit of the method(LOD is 0.084 mg/L),
#– The average was calculated between the two pooled vessels and vessel 3, with a two times weight factor of the pooled vessels.

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

Potassium Ferricyanide
TWA conc. (mg/L)
 

Mean

Std. Dev.

n

%Inhibition

Control

1.570

0.0298

6

 

0.042

1.582

0.0307

3

-0.77

0.26

1.469

0.0405

3

6.4#

0.78

1.359

0.2314

3

13*

3.0

0.337

0.1919

3

79*

9.6

0.000

0.0000

3

100*

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

Table 3          
Growth Rate and Percentage Inhibition at Different Time Intervals

 

Potassium Ferricyanide

TWA conc. (mg/L)

n

0 – 24h

24 – 48h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.516

 

1.530

 

1.663

 

0.042

3

1.630

-7.5

1.671

-9.2

1.445

13

0.26

3

1.203

21

1.583

-3.4

1.621

2.5

0.78

3

1.096

28

1.230

20

1.750

-5.2

3.0

3

0.659

57

0.321

79

0.030

98

9.6

3

0.187

88

-0.051

103

-0.135

108

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, Potassium Ferricyanide inhibited growth rate of this freshwater algae species significantly at TWA concentrations of 0.042 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.7 mg/L with a 95% confidence interval ranging from 1.4 to 2.0 mg/L.
The 72h-NOEC for growth rate inhibition was 0.042 mg/L. The 72h-EC10 for growth rate inhibition (ERC10) was 0.67 mg/L with a 95% confidence interval ranging from 0.50 to 0.90 mg/L.
Executive summary:

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

The Algae were exposed to a concentration range of 0.10, 0.32, 1.0, 3.2 and 10 mg/L and a blank control. The measured concentrations at the start of the test were at 107 – 111% of nominal, except for the lowest test concentration which was below the limit of detection (LOD). During the exposure period, the concentrations decreased to 38 – 88% of initial at the end of the test. Time weight average concentrations were calculated be 0.042, 0.26, 0.78, 3.0, 9.6 mg/L. Inhibition of growth rates increased with increasing concentrations of Potassium Ferricyanide from 0.26 mg/L upwards resulting in 100% inhibition at the highest test concentration. Statistically significant inhibition of growth rates was found at test concentrations of 0.26 mg/L and higher.

The 72h-NOEC for growth rate inhibition was 0.042 mg/L. The 72h-EC50for growth rate inhibition (ERC50) was 1.7 mg/L with a 95% confidence interval ranging from 1.4 to 2.0 mg/L.

The 72h-EC10 for growth rate inhibition (ERC10) was 0.67 mg/L with a 95% confidence interval ranging from 0.50 to 0.90 mg/L.

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

Description of key information

In a study with Raphidocelis subcapitata, Potassium Ferricyanide inhibited growth rate of this freshwater algae species significantly at TWA concentrations of 0.26 mg/L and higher. The 72h-EC50 for growth rate inhibition (ErC50) was 1.7 mg/L and the 72h-EC10 for growth rate inhibition (ErC10) was 0.67 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.7 mg/L
EC10 or NOEC for freshwater algae:
0.67 mg/L

Additional information

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

The Algae were exposed to a concentration range of 0.10, 0.32, 1.0, 3.2 and 10 mg/L and a blank control. The measured concentrations at the start of the test were at 107 – 111% of nominal, except for the lowest test concentration which was below the limit of detection (LOD). During the exposure period, the concentrations decreased to 38 – 88% of initial at the end of the test. Time weight average concentrations were calculated be 0.042, 0.26, 0.78, 3.0, 9.6 mg/L. Inhibition of growth rates increased with increasing concentrations of Potassium Ferricyanide from 0.26 mg/L upwards resulting in 100% inhibition at the highest test concentration. Statistically significant inhibition of growth rates was found at test concentrations of 0.26 mg/L and higher.

The 72h-NOEC for growth rate inhibition was 0.042 mg/L. The 72h-EC50for growth rate inhibition (ERC50) was 1.7 mg/L with a 95% confidence interval ranging from 1.4 to 2.0 mg/L. The 72h-EC10for growth rate inhibition (ERC10) was 0.67 mg/L with a 95% confidence interval ranging from 0.50 to 0.90 mg/L.

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