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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:
Experimental starting date: 8 June 2015. Experimental completion date: 14 March 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken at 0, 24, 48 and 72 h and the cell densitites determined for each contol and treatment group, using a Coulter® Multisizer Particle Counter. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormlaities recorded.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item was added to the surface of 2 L of culture medium to give 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirer using a stirring rate such that a vortex was formed to give a dimple at the ater surface. the stirring was stopped after 23 h and the mixture allowed to stand for 1 h. Visual observations made on the WAF indicated that a significant amount of dispered tes tiem was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and shoed there to be no micro-dispersions of test item present. The pH of the WAF was then measured and adjusted to pH 7.5. An aliquot of the pH adjusted WAF was inoculated with algal suspension to give the required test concentration of 100 mg/L loading rate WAF. The concentration and stability of aluminium in the test preparatopms was verified by chemical analysis at 0 and 72 h.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland

ACCLIMATION
- Culturing media and conditions (same as test or not): Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
pH adjusted WAF.
Nominal and measured concentrations:
100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Coonical flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass, 250 mL containing 100 mL of test preparation and plugged with polyurethan foam bungs and incubated at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380-730nm) and constantly shaken at approxiamtely 150 rpm for 72 h.
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 6.55 X10^5 cells per mL. Inoculation of 1 L of test medium with 7.6 mLof this algal suspension gave an initial nominal cell density of 5 x 10^3 mL and had no significant diltion effect on the final test concentration.
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Detailed composition: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl. NaNO3:25.5 mg/L; MgCl2.6H2O: 12.16 mg/L; CaCl2.2H2O: 4.41 mg/L; MgSO4.7H2O: 14.6 mg/L; K2HPO4: 1.044 mg/L; NaHCO3: 15.0 mg/L; H3BO3: 0.186 mg/L; MnCl2.4H2O: 0.415 mg/L; ZnCl2: 0.00327 mg/L; FeCl3.6H2O: 0.160 mg/L; CoCl2.6H2O: 0.00143 mg/L; Na2MoO4.2H2O: 0.00726 mg/L; =CuCl2.2H2O: 0.000012 mg/L; Na2EDTA.2H2O: 0.30 mg/L;
- The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item. Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Nominal loading rates of 10 and 100 mg/L. 72 h exposure. A control group was maintained under identical conditions but not exposed to the test item.
- Results used to determine the conditions for the definitive study: Based on the results of the reange-finding test the following loading rates were assigned to the first experiment: 6.25, 12.5, 25, 50 and 100 mg/L. The results obtained showed that significant inhibition of yield occured at 6.25 mg/L loading rate WAF. Based on the first experiment, a second experiment was conducted using a test concentration range of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF. Inhibitory effects to exposure were observed at 32 and 100 mg/L loading rate WAF. In order to determine whether the inhibition of growth observed in the second experiment was due to chelation of essential cations, a thrid experiment was conducted using a modified test media whereby the hardness was increased from 15 mg/L as CaCO3 to 150 mg/L as CaCO3, as recommended in the OECD Guidance Document on Aquatic Testing of Difficult Substances and Mixtures. The third experiment was conducted with nominal loading rates of 10 and 199 mg/L for a period of 72 h. Inhibitory effects to exposure were observed at 100 mg/L loading rate WAF. It had been noted during the range-finding test and initial experiments that the test item formed a strongly alkaline solution in test media which may have casued the inhibitory effects observed. As such, the definitive test was conducted with pH adjustment of the test preparation.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate.
Key result
Duration:
72 h
Dose descriptor:
other: EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: ErL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: ErL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: ErL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EyL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: EyL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Range-finding test: No significant effect on gorwth at 10 mg/L loadin rate WAF. Growth was observed to be resuced at 100 mg/L loading rate WAF. Chemical analysis of the test preparations at 0 h showed measured aluminium concentrations of 0.064 and 0.070 mg/L respectively were obtained whilst concentrations on 0.052 and 0.050 mg/L respectively were obtained at 72 h.
- Chemical analysis of test loading rates: Chemical analysis of the 100 mg/L lading rate WAF test preparation at 0 h showed a measured aluminium concentration of 0.044 mg/L was obtained. A slight decline in the measured aluminimum concentration obtained was seen at 72 h to 0.032 mg/L. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results are based on nominal loading rates only.
- Growth data: The growth rate and yield of the algae were not affected by the presence of the test item over the 72 h exposure period. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.
- Observation of abnormalities (for algal test): All test and control cultures were inspected at 72 h. After 72 h there were no abnormalities detected in the control or test cultures.
- Water quality criteria: The pH of the control cultures was observed to range from pH 7.4 at 0 h to pH 7.6 at 72 h. The pH deviation in to control cultures was less than 1.5 pH units after 72 h and therefore was within the limits given in the Test Guidelines.
- Vortex depth measurements: the vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.
- Observations on test item solubility: Observations on the test media were carried out during the mixing and testing of the WAF. At both the start and end of mixing, and following a 1 h standing period, the 100 mg/L laoding rate WAF was observed to have formed a clear colourless media column with solid particles of test tiem dispersed throughout the media column and floating at the media surface. As visual observations made showed there to be dispersed test item present it was considered appropriate to remove the WAF via filtration through a glass wool plug. Microscopic examination of the WAF after filtration showed there to be no micro-dispersions of test item present. At the start of the test all control and test coultures were observed to be clear colourless solutions. After the 72 h test period all control and test cultures were observed to be green dispersions.
Reported statistics and error estimates:
- Inibition growth rate: Statisitcal anlaytis of the gorwth rate data was carried out for the control and 100 mg/L loading rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences (P≥0.05), between the control and 100 mg/L loading rate WAF test group and therefore the "No Observed Effecr Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate WAF.
- Inhibition of yield: Statistical analysis of the yield data was carried out for the control and 100 mg/L loading rate WAF test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance. There was no statistically significant differences between the control and 100 mg/L loading rate WAF (P≥0.05) and, therefore the "No Observed Effecr Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate WAF.

Cell Densities and percentage inhibition of growth from the range-finding test

Nominal Loading Rate (mg/L)

Cell Densities* (cells per mL)

Inhibition values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

8.35E+03

1.14E+06

-

-

R2

7.01E+03

1.49E+06

Mean

7.68E+03

1.31E+06

10

R1

6.02E+03

1.10E+06

0

19

R2

6.66E+03

1.02E+06

Mean

6.34E+03

1.06E+06

100

R1

6.77E+03

3.03E+05

30

81

R2

6.54E+03

1.96E+05

Mean

6.66E+03

2.50E+05

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

R1and R2= Replicates 1 and 2

Cell densities and pH values in the definitive test

Nominal Loading Rate (mg/L)

pH

Cell Densities* (cells per mL)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.4

6.74E+03

2.59E+04

1.75E+05

8.14E+05

7.6

R2

5.68E+03

2.21E+04

1.81E+05

1.01E+06

R3

4.94E+03

2.44E+04

1.76E+05

6.99E+05

R4

6.35E+03

2.00E+04

2.17E+05

8.17E+05

R5

4.51E+03

3.03E+04

2.14E+05

9.86E+05

R6

4.70E+03

1.76E+04

1.84E+05

8.11E+05

Mean

5.49E+03

2.34E+04

1.91E+05

8.56E+05

100

R1

7.6

5.38E+03

2.49E+04

1.95E+05

9.03E+05

7.7

R2

4.74E+03

2.50E+04

2.27E+05

8.27E+05

R3

5.26E+03

3.34E+04

2.60E+05

9.74E+05

R4

6.19E+03

2.96E+04

1.83E+05

8.52E+05

R5

6.24E+03

2.45E+04

1.96E+05

7.71E+05

R6

4.37E+03

2.59E+04

1.85E+05

9.68E+05

Mean

5.36E+03

2.72E+04

2.08E+05

8.82E+05

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

R1– R6= Replicates 1 to 6

 

Daily specific growth rates for the control cultures in the definitive test

 

Daily Specific Growth Rate (cells/mL/hour)

Day 0 – 1

Day 1 – 2

Day 2 – 3

Control

R1

0.069

0.080

0.064

R2

0.062

0.088

0.072

R3

0.066

0.082

0.057

R4

0.058

0.099

0.055

R5

0.075

0.081

0.064

R6

0.052

0.098

0.062

Mean

0.064

0.088

0.062

 R1– R6= Replicates 1 to 6

 

Inhibition of growth rate and yield in the definitive test

Nominal Loading Rate (mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.071

 

8.07E+05

 

R2

0.074

 

1.00E+06

 

R3

0.069

 

6.94E+05

 

R4

0.071

-

8.11E+05

-

R5

0.073

 

9.82E+05

 

R6

0.071

 

8.06E+05

 

Mean

0.072

 

8.50E+05

 

SD

0.002

 

1.18E+05

 

100

R1

0.072

0

8.97E+05

 

R2

0.071

1

8.22E+05

 

R3

0.073

[1]

9.96E+05

 

R4

0.071

12

8.46E+05

 

R5

0.070

3

7.65E+05

 

R6

0.073

[1]

9.63E+05

 

Mean

0.072

1

8.77E+05

[3]

SD

0.001

 

8.12E+04

 

*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:
yes
Conclusions:
The short-term toxicity of the test item on the growth of Pseudokirchneriella subcapitata was investgiated in accordance with OECD Guideline 201 and, when exposed to a pH adjusted test solution, gave EL50 values of greater than 100 mg/L loadting rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

The short-term toxicity of the test item on the growth of Pseudokirchneriella subcapitata was investgiated in accordance with OECD Guideline 201 and, when exposed to a pH adjusted test solution, gave EL50 values of greater than 100 mg/L loadting rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

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