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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:
21 January - 21 February 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according with OECD Guideline No. 201 and in accordance with GLP principles. The reliability of the study is lower as there are non-biological effects (i.e. coloration, precipitation in the test medium) seen in the test system which cannot be fully explained and are not observed in the fish and daphnia tests. The test system of the algae study seems to induce specific conditions which might influence the stability of the ferrocyanide complex (e.g. test medium composition and light intensity). The analytical method used was based on Fe (recalculation of the Fe measurements to test substance was performed to obtain the average exposure concentrations) and it was shown to have a large margin of error for the lower test concentrations. Based on the fact that both the NOEC and EC50 were in the lower concentration range, the accuracy of these values is not high. However, based on the procedural recovery on March 4th 2013 of <83% at a nominal concentration of 0.1 mg/L, one can view the results as an underestimation of toxicity and thus the NOEC and EC50 can be viewed as worst case approach values for toxicity. Based on the non-biological effects seen in the test system and the decrease of Fe concentrations throughout the test period, one can be doubtful to which substance the algae are being exposed to.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2009)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
(2004)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Nominal concentrations
Range-finding test: 0.1 and 100 mg/L (for the highest test concentration samples from vessels with and without algae were collected)
Final test: 0 (blank-control), 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L (for the highest test concentration samples from vessels with and without algae were collected)
- Sampling: 9.6 ml of test solution were sampled from the approximate centre of the test vessels at t=0 h, t=24 h and t=72 h. At the end of the exposure period the replicates with algae were pooled at each concentration before sampling, except for the replicates of nominal 1.0 mg/L where one replicate was kept separately.
- Sample storage conditions before analysis: Samples were stored in the freezer (≤ -15°C) until the day of analysis. On the day of analysis, the samples were defrosted at room temperature. Nitric acid was added to the test samples to obtain a 4% Nitric acid matrix, after which the samples were analysed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with a nominal concentration of 100 mg/L for the range-finding test and 32 mg/L for the final test applying a short period of vigorous shaking to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 100 or 32 mg/L concentration, respectively, in test medium.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material: The final test solutions were all clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green microalgae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. M1 culture medium formulated according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) was used.The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2; according to the OECD 201 Guideline) at a cell density of 1 x 10^4 cells/ml.
- Culturing media and conditions: The pre-culture was maintained under the same conditions as used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
- Final test
At the start: 21.4°C
During the exposure period: 21.9 - 22.5°C.
pH:
- Final test
At the start: 8.1 - 8.3
At the end: 8.1 - 8.2
Nominal and measured concentrations:
- Nominal concentrations:
Range-finding test: 0 (blank-control), 0.10, 1, 10 and 100 mg/L
Final test: 0 (blank-control), 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L

- Measured concentrations: See the "Remarks on results including figures and tables" field for details. Samples were quantitatively analysed on Fe in water. Subsequently, test substance concentrations were calculated from these Fe-analyses.
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass, 100 ml capacity and containing 50 ml of test solution
- Type: closed
- Aeration: No
- Initial cells density: 1 x 10^4 cells/ml
- Control end cells density (final test): 128 x 10^4 cells/ml
- No. of vessels per concentration in the final test (replicates): 3
- No. of vessels per control in the final test (replicates): 6
- Other replicates (final test): 1 extra replicate of each test concentration and the control for sampling purposes; 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water purified by reverse osmosis
- Culture medium different from test medium: No. Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2)
- Intervals of water quality measurement: The pH was measured at the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuously
- Light intensity and quality: 97 to 107 µE.m-2.s-1; TLD-lamps of the type ‘Cool-white’ (30 Watt)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. At 24, 48 and 72 h, the cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (path length =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval. At the end of the test period nominal concentrations of 3.2 mg/L and higher were observed to contain test substance particles which might disturb spectrophotometric measurement. Therefore, algal density was also determined by use of a microscope and a counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations (final test): approx. 3
- Range finding study test concentrations: 0 (blank-control), 0.10, 1, 10 and 100 mg/L
- 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.:
0.05 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
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: CL 95%: 0.034-0.58
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.77-12 mg/L
Details on results:
- Range-finding test: From 24 hours of exposure onwards, test solutions containing the two highest test concentrations were observed to have a yellow/green colour. After 72 hours of exposure a slightly yellow colouring was also observed at nominal 1.0 mg/L. Based on the measured cell densities samples taken from nominal 0.1 and 100 mg/L were analysed.

- Final test: After 24 hours of exposure, test solutions containing the highest test concentration (27 mg/L) were observed to have a slightly yellow colour that intensified to yellow from 48 hours of exposure onwards. From 48 hours of exposure, the 5.8 mg/L concentration was observed to be slightly yellow. After 72 hours of exposure the three highest test concentrations contained undissolved particles and precipitation was observed.
Determination of the algal density by use of a microscope and a counting chamber at the end of the test showed that cell density did indeed decrease with increasing concentrations. Therefore, the spectrophotometric measurements performed at the end of the test could be used for the calculation of effect parameters.
Inhibition of yield increased with increasing concentration of Sodium ferrocyanide from 0.051 mg/L upwards resulting in >90% inhibition at and above 2.0 mg/L. The EC50 for yield inhibition (72h-EyC50) was 0.41 mg/L with a 95% confidence interval ranging from 0.16 to 1.1 mg/L. The 72h-EyC10 was 0.089 mg/L with a 95% confidence interval ranging from 0.033 to 0.24 mg/L.The 72h-NOEC for yield inhibition was 0.051 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

- Acceptability of the test:
1. In the control, cell density increased by an average factor of >16 within two days.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.
Results with reference substance (positive control):
- Potassium dichromate (K2Cr2O7): The EC50 for growth rate reduction (ErC50: 0-72h) was 0.97 mg/L with a 95% confidence interval ranging from 0.62 to 1.5 mg/L.
Reported statistics and error estimates:
- Determination of the average exposure concentrations:
The average exposure concentrations were calculated as: (24x√(Ct=0xCt=24)+48x√(Ct=24xCt=72))/ 72, being the Time Weight Average (TWA) of the concentrations of Sodium ferrocyanide measured in the samples taken at the start (Ct=0), after 24 hours (Ct=24) and the end of the test (Ct=72).

In case concentrations measured after 24 and or 72 hours of exposure were below the lowest calibration solution, the final exposure concentration(s) were taken as a factor of 2 below the applicable limit. This procedure is based on the OECD “Guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment”.

- Determination of the NOEC and calculation of the EC50:
For determination of the NOEC and the EC50 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 reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-Test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

- Range-finding test:

Percentage reduction of growth rate and inhibition of yield during the combined limit/range-finding test

Nominal conc.

Mean growth rate

Yield (0-72 h)

Sodium ferrocyanide

 

 

 

 

(mg/L)

µ (0-72 h)

Reduction (%)

x104cells/ml

Inhibition (%)

control

0.06686

122.54

0.10

0.06120

8.5

81.80

33.2

1.0

0.03630

45.7

13.66

88.9

10

0.02338

65.0

4.54

96.3

100

0.00196

97.1

0.18

99.9

- Final test:

Percentage reduction of growth rate (total test period) and percentage inhibition of yield

Average conc.

Mean growth rate

Yield (0-72 h)

Sodium ferrocyanide

 

 

 

 

(mg/L)

µ (0-72 h)

Reduction (%)

x104cells/ml

Inhibition (%)

control

0.06735

127.03

<0.0829

0.06595

2.1

114.67

9.7

0.051

0.06803

-1.0

133.03

-4.7

0.51

0.05540

17.7

56.24

55.7

2.0

0.03311

50.8

10.66

91.6

5.8

0.02571

61.8

5.45

95.7

27

0.01674

75.1

2.36

98.1

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Sodium ferrocyanide reduced growth rate and inhibited the yield of this fresh water algae species significantly at average exposure concentrations of 0.51 mg/L and higher. The EC50 for growth rate reduction (72h-ErC50) was 3.1 mg/L with a 95% confidence interval ranging from 0.77 to 12 mg/L. The EC50 for yield inhibition (72h-EyC50) was 0.41 mg/L with a 95% confidence interval ranging from 0.16 to 1.1 mg/L. The 72h-NOEC for both growth rate reduction and yield inhibition was 0.051 mg/L.
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Sodium ferrocyanide under static conditions at the following nominal concentrations: 0 (blank-control), 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L.

The EC50 for growth rate reduction (72h-ErC50) was 3.1 mg/L (95% confidence interval ranging from 0.77 to 12 mg/L) based on average exposure concentrations. The 72h-NOEC for growth rate reduction was 0.05 mg/L. The study is considered to be reliable with restrictions.

Description of key information

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the read across source substance sodium ferrocyanide under static conditions. The EC50 for growth rate reduction (72h-ErC50) was 3.1 mg/L (95% confidence interval ranging from 0.77 to 12 mg/L) based on average exposure concentrations. The 72h-NOEC for growth rate reduction was 0.05 mg/L. The study is considered to be reliable with restrictions based on the non-biological effects seen in the test system and the decrease of Fe concentrations throughout the test period, one can be doubtful to which substance the algae are actually being exposed to.

Key value for chemical safety assessment

Additional information

Key study (read across):

In a 72 h toxicity study conducted according to OECD guideline 201, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Sodium ferrocyanide under static conditions at the following nominal concentrations: 0 (blank-control), 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L.

The EC50 for growth rate reduction (72h-ErC50) was 3.1 mg/L (95% confidence interval ranging from 0.77 to 12 mg/L) based on average exposure concentrations. The 72h-NOEC for growth rate reduction was 0.05 mg/L. The study is considered to be reliable with restrictions.

The reliability of the study is lower as there are non-biological effects (i. e. coloration, precipitation in the test medium) seen in the test system which cannot be fully explained and are not observed in the fish and daphnia tests. The test system of the algae study seems to induce specific conditions which might influence the stability of the ferrocyanide complex (e. g. test medium composition and light intensity). The analytical method used was based on Fe (recalculation of the Fe measurements to test substance was performed to obtain the average exposure concentrations) and it was shown to have a large margin of error for the lower test concentrations. Based on the fact that both the NOEC and EC50 were in the lower concentration range, the accuracy of these values is not high. However, based on the procedural recovery on March 4th 2013 of <83% at a nominal concentration of 0.1 mg/L, one can view the results as an underestimation of toxicity and thus the NOEC and EC50 can be viewed as worst case approach values for toxicity. Based on the non-biological effects seen in the test system and the decrease of Fe concentrations throughout the test period, one can be doubtful to which substance the algae are being exposed to. The results are therefore difficult to interpret.

Supporting information (Tscheu-Schluter, 1983)

The publication does not contain enough information on the test system, conditions and organisms. The results are therefore difficult to interpret. The results show a high toxicity to algae. This high toxicity could be well attributed to the long exposure time (10 days instead of 3 days). This longer exposure time under light-dark conditions might trigger the formation of free cyanides, leading to an increased toxicity.

Supporting information (Pablo, et al. 1997)

Data have been obtained on the inhibition of cell division of the sensitive diatom, Nitzschia closterium, by potassium ferrocyanide. The results were consistent with free cyanide being the toxic component in the test solutions. The 72h-NOErC and the 72h-ErC50 for potassium ferrocyanide were determined to be 31 and 267 µg total cyanide/L, respectively. These values were recalculated to 0.09 and 1.62 mg test item/L.