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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
- Concentrations: 0, 62.5, 125, 250, 500 and 1000 mg/l nominal WAF loading rate (0, 10, 100 and 1000 mg/l nominal WAF loading rate in the range-finding study)
- Sampling method: Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours. Duplicate samples were taken and stored at -20°C for further analysis if necessary.
- Sample storage conditions before analysis: Not reported
Vehicle:
no
Details on test solutions:
PREPARATION OF CULTURE MEDIUM
Refer to Table 1. 15 ml of solution 1, 0.5 ml of solution 2 and 1.0 ml of solution 3 were mixed together and the volume adjusted to 1 litre with natural seawater (sterilised by membrane filtration, mean pore diameter 0.2 µm, prior to use). The pH of the culture medium was adjusted to 8.0 ± 0.2 by addition of dilute HCl or NaOH.

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Amounts of test material (125, 250, 500, 1000 and 2000 mg) were each seperately added to the surface of 2 litres of culture media via disposable plastic syringes to give loading rates of 62.5, 125, 250, 500 and 1000 mg/l, respectively. The test material was then stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. Stirring was conducted for 16 hours. The mixture was then allowed to stand for 4 hours. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel to a depth of 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 62.5, 125, 250, 500 and 1000 mg/l loading rate WAF.
- Evidence of undissolved material: Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: Diatom
- Strain: CCAP 1077/5
- Source: Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): In log-phase growth


ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions: Cultures were maintained in the laboratory by periodic replenishment of culture media descibed in Table 1 at a temperature of 20 ± 1°C under continuous illumination (approximately 7000 lux).
- Any deformed or abnormal cells observed: Not reported
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not relevant
Test temperature:
20 ± 1°C
pH:
Refere to Table 2
Dissolved oxygen:
Not reported
Salinity:
Not reported
Nominal and measured concentrations:
Nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass conical flask containing 100 ml of test preparation, plugged with polyurethane foam bungs and incubated in an INFORS Multitron(R) Version 2 Incubator
- Renewal rate of test solution: Test material was not renewed
- Initial cells density: 100000 cells/ml
- Control end cells density: 488000 cells/ml
- No. of vessels per concentration (replicates): Three (Two in the range-finding study)
- No. of vessels per control (replicates): Three (Two in the range-finding study)
- No. of vessels per vehicle control (replicates): SIx


GROWTH MEDIUM
- Detailed composition if non-standard medium was used: Refer to Table 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural sea water
- Total organic carbon: Refer to Table 3


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Culture media was adjusted to pH 8 ± 0.2 by the addition of dilute HCl or NaOH
- Photoperiod: Continuous illumination
- Light intensity: Approximately 7000 lux
- Salinity: Not reported


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Haemocytometer and light microscope


TEST CONCENTRATIONS
- Test concentrations: 0, 62.5, 125, 250, 500 and 1000 mg/l nominal WAF loading rate
- Range finding study
- Test concentrations: 0, 10, 100 and 1000 mg/l nominal WAF loading rate
- Results used to determine the conditions for the definitive study: No significant effects on growth were noted at 10 and 100 mg/l nominal WAF loading rate, growth was reduced at 1000 mg/l nominal WAF loading rate
Reference substance (positive control):
yes
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
125 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rate
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 210-300 mg/l nominal WAF loading rate (Calculated by the Litchfield and Wilcoxon method)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1 300 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF loading rate
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to derive 95% CL as the data generated does not fit the models available for the calculation of confidence limits
Details on results:
- Exponential growth in the control: Yes
- Observation of abnormalities: No abmormalities were detected in the control or test cultures
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Microscopic examination showed that there were no globules or micro-dispersions of test material
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- Results: Refer to Table 5
Reported statistics and error estimates:
Statistical analysis of the are under the growth curve data was carried out using one-way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences between the control, 62.5 and 125 mg/l WAF loading rates, however, all other loading rates were statistically significant. Therefore 125 mg/l WAF loading rate was identified as the NOELR.

Table 4: Effects of Cardolite NC-603 (technical grade) on the growth of Skeletonema costatum

Nominal loading rate

Replicate

Cell density (cells per ml)

AUC at 72 hours

% Inhibition

Growth rate (0-72 hours)

% Inhibition

0 hours

24 hours

48 hours

72 hours

Control

1

1.17 x104

2.67 x104

1.77 x105

5.10 x105

9.85 x106

-

0.052

-

2

1.17 x104

2.50 x104

1.57 x105

4.63 x105

3

1.00 x104

2.84 x104

1.70 x105

4.90 x105

Mean

1.11 x104

2.67 x104

1.68 x105

4.88 x105

62.5

1

1.17 x104

2.67 x104

1.97 x105

5.13 x105

1.02 x107

[4]

0.052

0

2

1.17 x104

2.84 x104

1.67 x105

5.00 x105

3

1.17 x104

2.50 x104

1.70 x105

4.93 x105

Mean

1.17 x104

2.67 x104

1.78 x105

5.02 x105

125

1

1.17 x104

2.17 x104

1.50 x105

5.03 x105

9.4 x106

5

0.053

[2]

2

1.17 x104

2.00 x104

1.47 x105

4.97 x105

3

1.00 x104

1.84 x104

1.63 x105

4.77 x105

Mean

1.11 x104

2.00 x104

1.53 x105

4.92 x105

250

1

1.00 x104

1.34 x104

3.34 x104

3.43 x105

4.74 x106

52

0.049

6

2

1.00 x104

1.34 x104

3.67 x104

3.60 x105

3

1.17 x104

1.50 x104

3.17 x104

3.53 x105

Mean

1.06 x104

1.39 x104

3.39 x104

3.52 x105

500

1

1.34 x104

1.00 x104

2.50 x104

1.97 x105

2.49 x106

75

0.039

25

2

1.34 x104

1.00 x104

3.17 x104

1.90 x105

3

8.35 x103

1.17 x104

2.50 x104

1.83 x105

Mean

1.17 x104

1.06 x104

2.72 x104

1.90 x105

1000

1

1.34 x104

1.17 x104

1.84 x104

9.33 x104

1.2 x106

88

0.032

38

2

8.35 x103

1.34 x104

1.50 x104

9.67 x104

3

8.35 x103

1.00 x104

1.50 x104

9.33 x104

Mean

1.00 x104

1.17 x104

1.61 x104

9.44 x104

AUC: Area under the curve

[Increased growth compared to the control]

Table 5: Effects of positive control on the growth of Skeletonema costatum

Nominal loading rate

Replicate

Cell density (cells per ml)

AUC at 72 hours

% Inhibition

Growth rate (0-72 hours)

% Inhibition

0 hours

24 hours

48 hours

72 hours

Control

1

1.17 x104

3.17 x104

1.20 x105

3.80 x105

7.62 x106

-

0.050

-

2

1.17 x104

3.00 x104

1.30 x105

3.70 x105

3

8.35 x103

3.00 x104

1.17 x105

3.97 x105

Mean

1.06 x104

3.06 x104

1.22 x105

3.82 x105

1.5

1

1.17 x104

1.34 x104

3.34 x104

1.43 x105

2.24 x106

71

0.036

28

2

1.17 x104

1.34 x104

3.17 x104

1.43 x105

3

8.35 x103

1.34 x104

3.17 x104

1.40 x105

4

1.00 x104

1.50 x104

3.00 x104

1.57 x105

5

1.17 x104

1.34 x104

3.67 x104

1.47 x105

6

1.00 x104

1.50 x104

3.50 x104

1.43 x105

Mean

1.06 x104

1.39 x104

3.31 x104

1.46 x105

AUC: Area under the curve

Comparison of areas under the growth curves

The area under the curve is taken to be an index of growth and was calculated using the following formula:

A=N1- N0x t1+N1+ N2– 2N0x (t2- t1) +Nn-1 + Nn – 2N0x (tn– tn-1)

         2                      2                                          2

where

A = area

N0 = cell concentration at the start of the test

N1 = cell concentration at t1

Nn = cell concentration at tn

t1 = time of first measurement (hours from start)

tn = time at nth measurement (hours from start)

Percentage inhibition of growth at each loading rate (IA) was calculated by comparing the area under the test curve (At) with that under the control curve (Ac) using the following equation:

IA =Ac- Atx 100

         Ac

Comparison of growth rates

The average maximum growth rate (µ) for each culture was calculated from the straight section of the growth curve using the following equation:

µ =lnNn- lnN1

         tn- t1

where

µ = average maximum growth rate

N0 = cell concentration at the start of test

Nn = cell concentration at tn

t0 = time at first measurement (0 hours)

tn = time of nth measurement (hours from the start)

Percentage inhibition of growth rate (Iµ) was calculated by comparing the growth rate of the test curve (µt) with that of the control curve (µc) using the following equation:

Iµ=µc- µtx 100

        µc

Executive summary:

In a 72 hour toxicity study, the cultures of Diatom (Skeletonema costatumCCAP 1077/5) were exposed to Cardolite NC-603 (technical grade) at nominal concentrations of 0, 62.5, 125, 250, 500 and 100 mg/l nominal WAF loading rate under static in accordance with the guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum). The EL50based biomass was 250 mg/l and the EL50based on growth rate was 1300 mg/l. There were no compound related phytotoxic effects.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for a marine algal growth inhibition study.

Results Synopsis

 

Test Organism: Skelonema costatum CCAP 1077/5

Test Type: Static

 

72 hr EC50 (biomass): 250 mg/l WAF loading rate        95% C.I.: 210 to 300 mg/l WAF loading rate

72 hr EC50 (growth rate): 1300 mg/l WAF loading rate

Description of key information

Key value for chemical safety assessment

Additional information

In a 72 hour toxicity study, the cultures of Diatom (Skeletonema costatum CCAP 1077/5) were exposed to Cardolite NC-603 (Technical Grade) at nominal concentrations of 0, 62.5, 125, 250, 500 and 100 mg/l nominal WAF loading rate under static in accordance with the guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum).


 


The EL50based biomass was 250 mg/l and the EL50based on growth rate was 1300 mg/l. There were no compound related phytotoxic effects.


 


The study is classified as Klimisch Code 2 (reliable with restriction) as it was performed to an internationally recognised method (ISO 10253) and was conducted to Good Laboratory Practice.


 

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