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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Test procedures followed those of US EPA Algal Assay Procedure: Bottle test (1971).
GLP compliance:
no
Analytical monitoring:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Source: U.S. E.P.A’s Environmental Research Laboratory, Corvallis, Oregon USA
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 ± 1 °C
pH:
See table 1
Nominal and measured concentrations:
56, 100, 320, 560 and 1000 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 125 ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3




OTHER TEST CONDITIONS
- Light intensity and quality: 4000 lux



EFFECT PARAMETERS MEASURED : Phytotoxicity (decrease in in vivo chlorophyll)
- Determination of cell concentrations: Cell counts were made using a hemacytometer and a Zeiss standard 14 compound microscope
- Chlorophyll measurement: Measurements of in vivo chlorophyll in cultures were performed using a Turner Model 111 flurometer


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Unknown
- Results used to determine the conditions for the definitive study: Concentrations for the definitive test were based on the results of a 96 hour range finding study
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: in vivo chlorophyll
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: in vivo chlorophyll
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
872
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: in vivo chlorophyll
Remarks on result:
other: 95% CL 313 - 2428
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
712 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: in vivo chlorophyll
Remarks on result:
other: 95% CL: 424-1198
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
655 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL: 439-977
Reported statistics and error estimates:
Each test concentration was converted to a logarithm and the corresponding percentage decrease of on vivo chlorophyll α or cell numbers was converted to a probit (Finney 1971), The 24, 48, 72 and 98 h EC50s and 95% confidence limits were then calculated by linear regression.

Table 1: pH values taken during test

Concentration (mg/l)

pH

0 h

96 h

Control

7.9

8.0

56

7.9

8.0

100

7.9

8.0

320

7.8

8.0

560

7.7

7.8

1000

7.6

7.8

Table 2: Percentage change in chlorophyll

Concentration (mg/l)

Percentage change

Chlorophyll alpha

Cell no

24h

48h

72h

96h

96h

Control

-

-

-

-

-

56

+7

+33

+2

+9

+3

100

+2

+33

+5

+8

+4

320

+2

+16

-19

-14

-15

560

-2

-3

-43

-42

-39

1000

-5

-16

-52

-66

-76

Exposure to cyanuric acid did not appear to adversely affect Selenastrum capricornutum until after 72 h of exposure. Growth of cultures exposed to concentrations <= 100 ppm was enhanced throughout the 96 h of exposure. After 96 h, decrease of in vivo chlorophyll alpha was from 14 % in cultures exposed to 320 ppm to 66 % in cultures exposed to 1000 ppm and decrease of cell numbers was from 15 % in cultures exposed to 320 ppm to 76 % in those exposed to 1000 ppm.The maximum test concentration was limited to 1000 ppm.

Conclusions:
Exposure to cyanuric acid did not appear to adveresely affect Selenastrum capricornutum until 72 hours of exposure. The estimated 24 hour and 48 hour EC50s based on decrease of in vivo chlorophyll alpha were >1000 ppm while the calculated 72- and 96-hour EC50s were 872 and 712 ppm respectively. Based on decrease of cell numbers the 96 hour calcualted EC50 was 655 ppm with 95% confidence limits of 439-977 ppm.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test material (15000 mg) was dispersed in culture medium with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 3 litres to give a 5000 mg/l stock dispersion. This stock dispersion was then filtered (0.2 µm Sartorius Sartopore, approximately 1 litre discarded to pre-condition the filter) to remove any undissolved test material and give a stock solution with a nominal concentration of 5000 mg/l. Serial dilutions were made from the stock solution to give further stock solutions of 2500, 1250, 625 and 313 mg/l, and an aliquot ( 1 litre) of each stock solution inoculated with algal suspension (5.0 ml) to give the required test concentrations.
Test organisms (species):
Navicula pelliculosa
Details on test organisms:
TEST ORGANISM
- Strain: SAG 1050-3
- Source (laboratory, culture collection): SAG: Collection of Algae Cultures Inst. Plant Physiology, University of Göttingen, Germany
- Method of cultivation: Cultures were maintained in the laboratory by the periodic replenishment of culture medium in the laboratory at a temperature of 21 ± 1 ºC under continuous illumination and aeration.


Test type:
static
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 ± 1ºC
pH:
The pH values of the control cultures were observed to decline from pH 7.5 at 0 hours to pH 7.1-7.2 at 96 hours. The pH deviation in the control cultures was less than 1.5 pH untis and therefore was within the limits of the test guideline. The test material vessels showed a concentration dependent increase in pH at 0 and 96 hours which was an effect of the test material (Refer to table 1 for individual measurements).
Nominal and measured concentrations:
313, 625, 1250, 2500 and 5000 mg/L (nominal)
314, 635, 1263, 2516, and 4978 mg/L (measured at 0 hours)
313, 628, 1245, 2466 and 4957 mg/L (measured at 96 hours)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed (foam bungs used to reduce evaporation)
- Material, size, headspace, fill volume: 250 mL glass conical flasks
- Initial cells density: 100000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: Continuous
- Light intensity and quality: 7000 lux


EFFECT PARAMETERS MEASURED : Growth rate and biomass
- Determination of cell concentrations: haemocytometer and light microscope



TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10 to 5000 mg/l
- Results used to determine the conditions for the definitive study: All test concentrations from the range finding study showed no significant inhibition of algal growth, therefore an initial test was conducted as a limit test at a concentration of 5000 mg/l The results of this test showed inhibition of algal growth. The definitive test was therefore conducted at test concentrations of 313, 625, 1250 and 5000 mg/l.
Duration:
72 h
Dose descriptor:
other: EbC50
Effect conc.:
2 700 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: equivalent to 2041 mg/L cyanuric acid
Duration:
96 h
Dose descriptor:
other: EbC50
Effect conc.:
> 5 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Equivalent to 3780 mg/l cyanuric acid
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 5 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Equivalent to 3780 mg/l cyanuric acid
Duration:
96 h
Dose descriptor:
other: ErC50
Effect conc.:
> 5 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Equivalent to 3780 mg/l cyanuric acid
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: equivalent to 945 mg/L cyanuric acid
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
5 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Equivalent to 3780 mg/l cyanuric acid
Details on results:
- Observation of abnormalities:All test and control cultures were inspected microscopically at 96 hours. THere were no abnormalities detected in any of the control or test cultures.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf, 1981) and Dennett’s multiple comparison procedure for comparing several treatments with a control (Dennett 1995) was carried out on the area under the growth curve and growth rate data to determine statistically significant differences between the test and control groups. All statistical analyses were performed using SAS computer software packages (SAS 1999 – 2001).

Table 1: pH measurements

Nominal concentration

(mg/L)

pH (0 h)

pH (96 h)

Control 1

7.5

7.2

Control 2

7.5

7.2

Control 3

7.5

7.1

Control Mean

7.5

7.2

313 R 1

8.3

7.5

313 R 2

8.3

7.5

313 R 3

8.3

7.5

313 Mean

8.3

7.5

625 R 1

8.6

7.6

625 R 2

8.6

7.6

625 R 3

8.6

7.6

625 Mean

8.6

7.6

1250 R1

8.9

7.8

1250 R 2

8.9

7.8

1250 R 3

8.9

7.8

1250 Mean

8.9

7.8

2500 R1

9.1

8.1

2500 R 2

9.1

8.1

2500 R 3

9.1

8.1

2500 Mean

9.1

8.1

5000 R 1

9.2

8.3

5000 R 2

9.2

8.3

5000 R 3

9.2

8.3

5000 Mean

9.2

8.3

Table 2: Cell densities

Nominal concentration

(mg/L)

Cell Densities* (cells per mL)

0 h

24 h

48 h

72 h

96 h

Control 1

1.17 x 104

4.84 x 104

9.17 x 104

1.92 x 105

1.17 x 106

Control 2

1.17 x 104

4.37 x 104

8.84 x 104

1.83 x 105

1.18 x 106

Control 3

1.00 x 104

4.34 x 104

9.67 x 104

1.62 x 105

1.09 x 106

Control Mean

1.11 x 104

4.62 x 104

9.23 x 104

1.79 x 105

1.15 x 106

313 R 1

1.34 x 104

5.50 x 104

1.03 x 105

1.82 x 105

1.10 x 106

313 R 2

1.17 x 104

4.84 x 104

1.23 x 105

1.85 x 105

1.25 x 106

313 R 3

1.17 x 104

5.00 x 104

9.67 x 104

1.70 x 105

1.18 x 106

313 Mean

1.23 x 104

5.11 x 104

1.08 x 105

1.79 x 105

1.18 x 106

625 R 1

1.00 x 104

3.50 x 104

7.17 x 104

1.90 x 105

1.25 x 106

625 R 2

1.34 x 104

5.00 x 104

6.84 x 104

1.73 x 105

1.23 x 106

625 R 3

1.17 x 104

4.50 x 104

8.67 x 104

1.92 x 105

1.13 x 106

625 Mean

1.17 x 104

4.33 x 104

7.56 x 104

1.85 x 105

1.20 x 106

1250 R1

1.17 x 104

3.50 x 104

7.50 x 104

1.80 x 105

1.16 x 106

1250 R 2

1.17 x 104

3.84 x 104

6.17 x 104

1.87 x 105

1.11 x 106

1250 R 3

1.34 x 104

4.00 x 104

7.34 x 104

1.63 x 105

1.12 x 106

1250 Mean

1.23 x 104

3.78 x 104

7.00 x 104

1.77 x 105

1.13 x 106

2500 R1

1.00 x 104

2.67 x 104

4.34 x 104

1.27 x 105

1.20 x 106

2500 R 2

1.00 x 104

2.67 x 104

3.50 x 104

9.50 x 104

1.04 x 106

2500 R 3

1.17 x 104

3.00 x 104

4.50 x 104

1.18 x 105

1.18 x 106

2500 Mean

1.06 x 104

2.78 x 104

4.11 x 104

1.13 x 105

1.14 x 106

5000 R 1

1.17 x 104

1.00 x 104

1.67 x 104

1.20 x 105

1.15 x 106

5000 R 2

1.17 x 104

1.17 x 104

1.50 x 104

1.23 x 105

1.18 x 106

5000 R 3

1.17 x 104

8.35 x 103

1.50 x 104

1.18 x 105

1.10 x 106

5000 Mean

1.17 x 104

1.00 x 104

1.56 x 104

1.20 x 105

1.14 x 106

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 fields of view for each of the replicate flasks

Validity criteria fulfilled:
yes
Conclusions:
The 72 h EbC50 value = 2700 mg/L and the 72 h ErC50 value > 5000 mg/L. The NOEC based on area under the curve was 625 mg/L; in terms of the 0-72 hour growth rate the NOEC was 1250 mg/L. After 96 hours exposure EC50 values of greater than 5000 mg/L were obtained. The NOEC based on area under the curve was 1250 mg/L; in terms of the 0-96 hour growth rate the NOEC was 5000 mg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from the control (replicates R1 - R6 pooled) and the 100 mg/L test group (repliacte R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 1 L to give 100 mg/L stock solution. This stock solution was inoculated with algal suspension (1.2 mL) to give the required test concentration of 100 mg/L
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 1077/5
- Source: Culture collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory , Oban, Argyll, Scotland
- Method of cultivation: Culture maintained in laboratory by periodic replenishment of culture medium and kept under constant agitation by an orbital shaker (100 – 150 rpm) and constant illumination at 20 ± 1°C


Test type:
static
Limit test:
yes
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
20 ± 1°C
pH:
pH was measured at 0 and 72 h. Refer to table 1
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
100 mg/L (equivalent to 76 mg/l cyanuric acid) (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flask
- Initial cells density: 3 x 10^3 cells per mL
- No. of vessels per concentration: 6
- No. of vessels per control: 6



OTHER TEST CONDITIONS
- Photoperiod: Constant illumination
- Light intensity and quality: Approximately 7000 lux provided by warm lighting (380 - 730nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Samples were taken at 0, 24, 48 and 72 hours and cells densities determined
- Determination of cell concentrations: haemocytometer and light microscope


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: The range finding study was conducted to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours
- Results used to determine the conditions for the definitive study: Based on the results of the range finding study a 'limit' test was conducted for the definitive test at a concentration of 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: equivalent to >76 mg/L cyanuric acid
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
biomass and growth rate
Basis for effect:
other:
Remarks on result:
other: equivalent to 76 mg/L cyanuric acid
Details on results:
- Observation of abnormalities: All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72 h test period all control and test cultures were observed to be very pale brown dispersions.
Results with reference substance (positive control):
- ErC50 (0-72 h): 3.3 mg/L; 95% confidence limits 2.9 - 3.7 mg/L
- EbC50 (0 -72 h): 1.4 mg/L; 95% confidence limits 1.2 - 1.6 mg/L
- NOEC (based on growth rate): 1.25 mg/L
- NOEC (based on biomass): 0.313 mg/L
Reported statistics and error estimates:
One way analysis of variance according to Bartletts test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnetts multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 – 2001). Where appropriate 95% confidence limit for EC50 values were calculated using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949)

Table 1: pH measurements for the definitive test

Nominal concentration

(mg/L)

pH (0 h)

pH (72 h)

Control                 R1

8.0

8.0

Control                 R2

8.0

8.0

Control                 R3

8.0

8.0

Control                 R4

8.1

8.0

Control                 R5

8.1

8.1

Control                 R6

8.1

8.1

Positive control   R1

7.9

7.9

Positive control   R2                            

8.0

8.0

Positive control   R3                  

8.0

8.0

100                        R1

8.0

8.1

100                        R2

8.0

8.1

100                        R3

8.0

8.1

100                        R4

8.0

8.1

100                        R5

8.1

8.1

100                        R6

8.1

8.1

 

Validity criteria fulfilled:
yes
Remarks:
The cell concentration of the control cultures increased by a factor of 28 after 72 hours. The pH deviation in the control cultures was less than 1.0 pH units after 72 hours and therefore was within the limits given in the Test Guidelines
Conclusions:
Exposure of Skeletonema costatum to cyanuric acid gave EC50 values of greater than 100 mg/L (equivalent to 76 mg/L cyanuric acid) and correspondingly the NOEC was 100 mg/L (equivalent to 76 mg/L cyanuric acid). Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Description of key information

Two algal growth inhibition studies have been performed for the monosodium salt of cyanuric acid  with freshwater algae (OECD 201) and one study with marine algae.

Key value for chemical safety assessment

EC50 for freshwater algae:
3 780 mg/L
EC50 for marine water algae:
76 mg/L
EC10 or NOEC for freshwater algae:
945 mg/L
EC10 or NOEC for marine water algae:
76 mg/L

Additional information

Freshwater algae Navicula pelliculosa were exposed to 313, 625, 1250, 2500 and 5000 mg/L (equivalent to 237, 473, 945, 1890 and 3780 mg/L cyanuric acid). The test concentration of 5000 mg/L was the highest attainable test concentration that could be prepared due to the solubility of the test material. After 96 h exposure EC50 values of > 5000 mg/L (equivalent to 3780 mg/L cyanuric acid) were obtained. The NOEC was 1250 mg/L (equivalent to 945 mg/L cyanuric acid)

In another study the freshwater algae Selenastrum capricornutum were exposed to 56, 100, 320, 560 and 1000 mg/L nominal concentrations of cyanuric acid. Exposure to cyanuric acid did not appear to adversely affect Selenastrum capricornutum until 72 hours of exposure. The estimated 24 hour and 48 hour EC50s based on decrease of in vivo chlorophyll alpha were >1000 ppm while the calculated 72- and 96-hour EC50s were 872 and 712 ppm respectively. Based on decrease of cell numbers the 96 hour calculated EC50 was 655 ppm with 95% confidence limits of 439-977 ppm.

In a limit test marine algae Skeletonema costatum were exposed to a nominal concentration of 100 mg/L to the monosodium salt of cyanuric acid. The EC50 value was >100 mg/L (equivalent to 76 mg/L cyanuric acid) and correspondingly the NOEC was 100 mg/L (equivalent to 76 mg/L cyanuric acid). Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.