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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:
5 december 2008 - 20 january 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Despite the lack of analytical monitoring data, it can be reasonably assumed that the test concentrations remained stable over the test duration from the analytical measurements reported in the long-term toxicity test to Daphnia magna performed on a structural analogue substance (Höger, 2013).
Qualifier:
according to guideline
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test solutions were prepared by dilution series from an initial 1000mg/L stock solution and without any organic solvent.
- Controls: treated seawater without test item.
- Reference vessels: prepared from a main stock of 100 mg/L of 3,5-dichlorophenol.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: Skeletonema costatum.
- Culture number: C5B2A.
- Method of cultivation: pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating treated seawater with nutrient medium (culture medium) to a cell density of approximately 2 x 10E3 to 10E4 cells per millilitre. The pre-cultures were incubated at approximately 20 +/- 2°C under constant illumination for 3 days +/- 1 day, and where uses as the inoculum source for the test.

ACCLIMATION
- Acclimation period: not specified.
- Culturing media and conditions: see method of cultivation above.
- Any deformed or abnormal cells observed: data not available.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
19.1 - 21.6 °C
pH:
Test item vessels: 8.13 - 8.18 (at 0h)
Control vessels: 8.11 - 9.10 (during the test)
Dissolved oxygen:
No data
Salinity:
36 ppt at 0h (in diluent water)
Nominal and measured concentrations:
Nominal concentrations: 10, 18, 32, 56 and 100 mg/L (test item)
Details on test conditions:
TEST SYSTEM
- Test vessel:
-- Type: borosilicate glass conical flasks;
-- Material, size, headspace, fill volume: 100-mL flasks, filled with 80mL of test medium seawater.
- Agitation: during the test, the test solutions were continuously stirred by orbital shaker.
- Renewal rate of test solution (frequency/flow rate): a static, non-renewal exposure system was used.
- Initial cells density: 10000 algal cells per mL of test medium.
- No. of vessels per concentration (replicates): 2.
- No. of vessels per control (replicates): 4.

TEST MEDIUM / WATER PARAMETERS
Culture medium was prepared from natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 0.2 µm. The filtered treated seawater was enriched with nutrients and vitamins in accordance with ISO Guidelines. The salinity of the enriched natural seawater at 0h was 36 °/oo +/- 4°/oo.

OTHER TEST CONDITIONS
- Sterile test conditions: yes.
- Adjustment of pH: at 0h for the test item concentration of 100 mg/L.
- Photoperiod: continuously illuminated.
- Light intensity and quality: the measured light intensity (7160 - 8900) was achieved by four 40W tubes mounted at a distance of approximatively 40 cm directly above the test area. Light intensity values were measured daily during the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counts of algal cell numbers were carried out daily by Fluorometer measurements. Three readings were performed on each test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not stated.
- Range finding study: yes.
- Test concentrations: 1 - 10 - 100 - 1000 mg/L (test item).
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 15.08-18.65 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 5.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
The following validity criteria were fulfilled:
- the biomass in the control cultures has increased exponentially by a factor greater than 16 within the 72-hour test period;
- test conditions (pH, temperature) were maintained during the test.

There is a lack of information of data relating to the analytical monitoring showing that the concentrations of the test item were maintained during the test. However, according to the long-term toxicity test to Daphnia performed on a very similar substance, the substance is stable during 48 hours. Thus, we can consider that in a short-term study, the substance is also stable.
Results with reference substance (positive control):
For evaluation of the quality of the aquatic organisms and the experimental conditions, 3,5-dichlorophenol was tested as a positive control.
72h EC50 = 2.19 mg/L (with 95% confidence limits : 2.03 - 2.34 mg/l)
(See results on tables 2 and 5 below)
Reported statistics and error estimates:
The raw data for each duplicate vessel and time period were averaged, to give values for each concentration of cell volume. Growth rate was calculated on the basis of these measurements. Daily intrinsic growth rate was calculated for each duplicate for each time period using an exponential model :
Nt = N0.ekt where :
Nt : volume or number at time t
N0 = volume or number at beginning of test
t = time in days
k = growth rate (d -1)

The average value of k for each time interval was calculated for each concentration. Since the criterion of effect was the concentration causing 50% reduction in growth rate with respect to the controls, the response for each concentration was estimated from . Effect = 1- (control k/treatment k).

The resulting values represent proportional reduction in growth rate. The EC50 for each time interval and the 72h EC90 and NOEC values were calculating using an appropriate statistical method from the ToxCalc Version 5 software.

Table 4 - Fluorescence measurements (ppb) in test vessels after 24, 48 and 72h

Concentration (mg/L)

24h

48h

72h

10a

0.161

3.414

34.907

10a

0.178

3.367

34.975

10a

0.135

3.351

34.867

10b

0.183

3.875

36.232

10b

0.200

3.879

36.273

10b

0.205

3.849

36.177

18a

0.033

0.219

3.383

18a

0.034

-

3.376

18a

0.045

-

3.341

18b

0.040

0.199

4.232

18b

0.046

-

4.157

18b

0.042

-

4.129

32a

0.058

0.011

0.172

32a

0.058

-

-

32a

0.060

-

-

32b

0.039

0.009

0.199

32b

0.040

-

-

32b

0.040

-

-

56a

0.028

0.018

0.472

56a

0.036

-

-

56a

0.022

-

-

56b

0.048

0.033

0.414

56b

0.005

-

-

56b

0.039

-

-

100a

0.016

0.014

0.113

100a

0.001

-

-

100a

0.014

-

-

100b

0.022

0.031

0.127

100b

0.016

-

-

100b

0.014

-

-

 

Table 5 - Fluorescence measurements (ppb) in control and referencesubstance (3,5 -DCP) after 72h

Replicate

72h

Rep growth

Mean growth

Measurement 1

Measurement 2

Measurement 3

Control 1

47.406

47.489

44.259

1.00

 

1.00

Control 2

44.461

44.858

45.081

1.03

Control 3

55.560

55.454

55.601

0.98

Control 4

49.561

49.809

49.812

0.99

1.0a

42.755

50.684

51.023

1.00

0.99

1.0b

46.574

46.256

46.355

0.99

1.8a

13.181

13.169

13.138

0.71

0.69

1.8b

11.148

11.116

11.037

0.67

3.2a

0.608

0.585

0.566

0.02

0.01

3.2b

0.473

0.479

0.478

0.00

 

Table - Coefficient of variation at 24, 48 and 72h (%)

24 h control CV %

7.59

48 h control CV %

1.53

72 h control CV %

2.10

Validity criteria fulfilled:
yes
Conclusions:
The test item exhibited adverse effects on Skeletonema costatum at nominal concentrations of 18 mg/L and higher. The 72h-EC50 (growth rate) value was determined to be 16.89 mg/L (corresponding to 5.1 mg active content/L) while the 72h-NOEC value was determined to be 10 mg/L (corresponding to 3 mg active content/L).
Executive summary:

The toxicity of the REACh substance (2-Hydroxy-3-sulphopropyl) dimethyl[3-[(1-oxododecyl) amino]propyl]ammonium hydroxide (EC 242-893-1) was investigated in a GLP-compliant study performed with the marine algal species Skeletonema costatum in accordance with ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum). The test item exhibited adverse effects on Skeletonema costatum at nominal concentrations of 18 mg/L and higher. The 72h-EC50 (growth rate) value was determined to be 16.89 mg/L (corresponding to 5.1 mg active content/L) while the 72h-NOEC value was determined to be 10 mg/L (corresponding to 3 mg active content/L).

Description of key information

The toxicity of the REACh substance (2-Hydroxy-3-sulphopropyl) dimethyl[3-[(1-oxododecyl) amino]propyl]ammonium hydroxide (EC 242-893-1) was investigated in a GLP-compliant study (Hudson, 2009) performed with the marine algal species Skeletonema costatum in accordance with ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum). The test item exhibited adverse effects on Skeletonema costatum at nominal concentrations of 18 mg/L and higher. The 72h-EC50 (growth rate) value was determined to be 16.89 mg/L (corresponding to 5.1 mg active content/L) while the 72h-NOEC value was determined to be 10 mg/L (corresponding to 3 mg active content/L).

NB. In case the solid content of the test item was not reported in a study report, ECx values were expressed in active content. If relevant, these values were used for the chemical safety assessment (worst-case values).

Key value for chemical safety assessment

EC50 for marine water algae:
5.1 mg/L
EC10 or NOEC for marine water algae:
3 mg/L

Additional information

The toxicity to aquatic (marine) algae of the substance EC 242-893-1 was investigated in a GLP-compliant study (Hudson, 2009) performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 2) and was selected as key study for the endpoint.

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