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Ecotoxicological information

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:
From November 01 to 09, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
not specified
Details on test solutions:
The following test flasks were set up:
• Test solution (Tn); containing test medium, test item and algae (three replicates per concentration)
• Blank control (Bn); containing test medium and algae (six replicates)
Because the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution. A 10 mg/l stock solution was prepared by adding the test item to sterile-filtered algal medium in sterile glassware and stirred for 5-10 minutes. The respective dilutions of this stock solution was then prepared with sterile-filtered algal medium (pH adjusted at 8.0 ± 0.2) and sterile glassware. 100 ml of each test solution was added to the sterile test flasksand inoculated with an exponentially growing pre-culture of algae.
Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls.
For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81 SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from axenic slope culture
Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
Temperature: 21 to 24 °C, maintained at ± 2 °C in a thermo-controlled room
Control of sensitivity: None – the culture is re-purchased annually
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 to 24°C, maintained at ± 2 °C in a thermo-controlled room
pH:
Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
Nominal and measured concentrations:
0.100, 0.0562, 0.0316, 0.0178 and 0.010 mg/l (nominal concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm)
- Initial cells density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)
- No. of vessels per concentration (replicates): 3
- No. of vessels per blank control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM
Test medium: Recommended OECD medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Illumination - Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
Light intensity : the light intensity amounts to about 3000 lux which corresponds to about 40 μE•m-2•s-2
Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: determined every 24 h using a spectrophotometer at 680 nm wavelength (Shimadzu UV 1800,Shimadzu Schweiz GmbH,Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.
- pH: determined in the medium batch which is used for the preparation of the test solutions, in each test solution, and in the blank control and the highest concentration at the end of the test.
- Temperature: determined with a data logger in an additional vessel in the incubation chamber several times per day
- Light: measured at the start and the end of the test at several points over the whole incubation area



TEST CONCENTRATIONS
Range finding study
- Test concentrations: 0.01, 0.1 and 1.0 mg/l
- Results used to determine the conditions for the definitive study: prior to the definitive test a first non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of INTERCURE 18 was performed. At all three concentrations, 100% inhibition was observed. Due to the high toxicity observed in the first non-GLP range finding test, a second non-GLP range finding test was performed with nominal concentrations of 0.01, 0.1 and 1.0 mg/l. At 0.01 mg/l no inhibition was observed; at 0.1 mg/l 86 % of inhibition was observed; at 1.0 mg/l 100 % of inhibition wa observed.
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.07 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.046 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 0.033 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 0.035 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.032 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.032 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass

Test concentrations

Due to the sensitivity of the HPLC method, which is too low in regard of theinvestigated test concentrations, only the stock solution exposed to the same conditions as the test vessels was measured. The concentration of INTERCURE 18 in the stock solution was measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. These analyses revealed that the concentration of the stock solution was stable over the 72 h test period (84 % of initial value) and it can be assumed, that the test concentrations were also stable during the whole test period.

Therefore, the effective concentrations ErCx/EyCxwere assessed based on the nominal concentrations of the test item.

Determination of the effect concentrations based on growth rate

The dose-response curve encompassed the range from no observed effects to 81 % inhibition, as compared to the blank controls.

The median effect concentration with respect to growth rate (ErC50/0–3 days) of INTERCURE 18 to Desmodesmus subspicatus was calculated to be 0.070 mg/l (95 % confidence limits: 0.064–0.076 mg/l).

The low effect concentration with respect to growth rate ErC10was calculated to be 0.033 mg/l (95 % confidence limits: 0.026–0.039 mg/l).

The no observed effect concentration (NOErC) with respect to growth rate was determined to be 0.032 mg/l.

Determination of the effect concentrations based on yield (biomass production)

The dose-response curve encompassed the range from no observed effects to 96 % inhibition, as compared to the blank controls.

The median effect concentration with respect to yield (EyC50/0–3 days) of INTERCURE 18 to Desmodesmus subspicatus was calculated to be 0.046 mg/l (95 % confidence limits: 0.044–0.048 mg/l).

The low effect concentration with respect to yield EyC10was calculated to be 0.035 mg/l (95 % confidence limits: 0.031–0.037 mg/l).

The no observed effect concentration (NOEyC) with respect to yield was determined to be 0.032mg/l.

Validity criteria fulfilled:
yes
Conclusions:
ErC50 (72h)= 0.070 mg/l
EyC50 (72h)= 0.046 mg/l
Executive summary:

Method

The effective concentrations (ErC10, ErC50and EyC10, EyC50) as well as the no observed effect concentrations (NOErC and NOEyC), of INTERCURE 18 to the green alga Desmodesmus subspicatus based on the average specific growth rate and the yield (algal biomass production) were investigated over a period of 72 h, according to the OECD guideline 201.

The test solutions were prepared by respective dilutions of a stock solution. The nominal concentrations were 0.100, 0.0562, 0.0316, 0.0178 and 0.010 mg/l. Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

Results

The 72 h ErC50and EyC50values of INTERCURE 18 on the green alga Desmodesmus subspicatus were 0.070 mg/l and 0.046 mg/l, respectively. These values are based on the nominal concentrations.

Description of key information

ErC50 (72h) = 0.070 mg/l

EyC50 (72h) = 0.046 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:
0.07 mg/L
EC10 or NOEC for freshwater algae:
0.032 mg/L

Additional information

The effective concentrations (ErC10, ErC50and EyC10, EyC50) as well as the no observed effect concentrations (NOErC and NOEyC), of INTERCURE 18 to the green Desmodesmus subspicatus based on the average specific growth rate and the yield (algal biomass production) were investigated over a period of 72 h, according to the OECD guideline 201.

The test solutions were prepared by respective dilutions of a stock solution. The nominal concentrations were 0.100, 0.0562, 0.0316, 0.0178 and 0.010 mg/l. Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

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