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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 29, 2001 till December 14, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD Guideline 201 (Algal Growth Inhibition Test) under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
according to OECD Guideline 201 (Alga, Growth Inhibition Test) with no deviations
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: ZP-TIX 1014
Batch No: NW-01-028
Expiration date: December 30, 2001
Purity: > 99%
Stability in water: Determined in RCC Study 818897
Solubility in water: Insoluble (according to the Sponsor Information) Determined in RCC study 818651 (<0.9mg/L)
Aggregate state/Physical form at room temperature: Solid
Color: White (Visually determined at RCC)
Storage conditions: At room temperature about 20°C, in the dark

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Small volumes of the test media and control (1.0 mL) were taken after 24, 48 and 72h exposure, and were not replace.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accomodated fractions (WAFs) with loading rates of 4.6, 10, 22, 46 and 100 mg/L test item were tested.
WAFs were prepared by dispersing 9.5, 11.0, 11.4, 24.2 and 29.7mg of test item in 2000, 1000, 500, 500 and 300mL test water
Ultrasonificated for 15 and intense stirring for 96h at RT in the dark.
The long stirring period were chosen to enrich the test water with the maximum concentration on degradation products
non dissolved fraction was filtered with 0.45µm filter
Filtrates were used as test media
Test media were prepared just before the start of the test

- Controls: test water without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): test water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): yes, non dissolved fraction was filtered with 0.45µm filter

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: Strain No. 86.81
- Source (laboratory, culture collection): "Sammlung von Algenkulturen Göttingen (SAG, Experimentelle Phykologie und Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, D-37073 Göttingen, Germany).
- Age of inoculum (at test initiation): test were started by inoculum of 10000 algal cells per mL test medium (0 hours)
these cells were taken from an exponentially growing pre-culture, which was set up 4 days
- Method of cultivation: cultivated and tested in synthetic test water, prepared according to the test guideline
- Stock culture: stock culture on solid agar at 8°C.

ACCLIMATION
- Acclimation period: 4 days prior to the test
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no data

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
The EC Values were calculted for both parameters, the algal biomass (b) and the groth rate (r), after 72 hours test duration. All results are based onloading rates.
Post exposure observation period:
none

Test conditions

Hardness:
0.24mmol/L (24mg/L) as CaCO3
Test temperature:
22 °C
pH:
7.9-8,9
Dissolved oxygen:
no data
Salinity:
Macro-nutrients:
NaHCO3 50.0mg/L
CaCl2 x2H2O 18.0mg/L
NH4Cl 15.0mg/L
MgSO4 x7H2O 15.0mg/L
MgCl2 x 6H2O 12.0mg/L
KH2PO4 1.6mg/L
Trace elements:
Na2EDTAx2H2O 100.0 µg/L
FeCl2 x6H2O 80.0 µg/L
MnCl2 x4H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4 x2H2O 7.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 x 6 H2O 1.5 µg/L
CuCl2 x 2 H2O 0.01µg/L
Nominal and measured concentrations:
Nominal concentration of the water accommodated fractions:
4,6, 10, 22, 46 and 100mg/L

The test item is a poorly soluble mixture of isocyanates and it is generally known, that these compounds degrade fast in water. Due to this characteristics, the test item could not be anayltically measured in the test medium.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open flasks (covered with a glass dish
- Material, size, headspace, fill volume: Erlenmeyer flask (glass)
- Aeration:
- Renewal rate of test solution (frequency/flow rate): static test
- Initial cells density: 10000 algal cells per mL
- Control end cells density: 48x10000 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Volume: 15 mL algal suspension for each replicate

GROWTH MEDIUM
- Standard medium used: yes
Macro Nutrients:
NaHCO3 50.0 mg/L
CaCl2 18.0mg/L
NH4CL 15.0mg/L
MgSO4 x7H2O 15.0 mg/L
KH2PO4 1.6mg/L
Trace elements:
Na2EDTAx2H2O 100 µg/L
FeCL3x6H2O 80.0 µg/L
MnCL2x4H2O 415.0 µg/L
H3BO3 185.0 µg/l
Na2MoO4x 2H2O 7.0 µg/L
ZnCL2 3.0 µg/L
CoCL2 x6H2O 1.5 µg/L
CuCL2x2H20 0.01 µg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- pH value: measured for each loading rate and control at the start and the end of the test
- Apperance: Apperance of test media was recorded daily.
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: see pH value
- Ca/mg ratio: See nutrition
- Conductivity: no data
- Intervals of water quality measurement: no data


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no data
- Photoperiod: continuously illuminated
- Light intensity and quality: light intensity 8200 Lux (mean value) range 7200 to 8700 Lux achieved by fluorescent tubes (Philips TLD 36W/840)
- Salinity (for marine algae): see above (nutrition)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: alagl density determined via electronic particle counter (Coulter Counter, Model ZM)
at least two measurements per sample
small volumes of the test media and controls (1.0mL) were taken

TEST CONCENTRATIONS
- Nominal concentration of the water accommodated fractions (WAFs):4.6, 10, 22, 46 and 100mg/L
- Justification for using less concentrations than requested by guideline: WAFs with loading rates in excess of 100mg/L were not tested in compliance with EU Commission Directive 92/69/EEC

The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water to obtain the following final nomination concentrations:
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accomodated fractions
Basis for effect:
biomass
Remarks on result:
other: this result is based on loading rates
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accommodated fractions
Basis for effect:
growth rate
Remarks:
4,6 mg/L
Remarks on result:
other: The result is based on loading rates
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accommodated fractions
Basis for effect:
biomass
Remarks on result:
other: This result is based on loading rates
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accommodated fractions
Basis for effect:
growth rate
Remarks on result:
other: This result is based on loading rates
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 78 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accommodated fractions
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits could not be determined
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accommodated fractions
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits could not be determined
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 5.5 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accommodated fractions
Basis for effect:
biomass
Remarks:
5,5 mg/L
Remarks on result:
other: 95% confidence limits could not be determined
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 9.1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Water accommodated fractions
Basis for effect:
growth rate
Remarks:
9,1 mg/L
Remarks on result:
other: 95% confidence limits could not be determined
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no obvious effects on the shape and size of the alga cells
- Effect concentrations exceeding solubility of substance in test medium: no remarkable observations were made concerning the test media. All test media were clear solutions throughout the test periode.
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
EC10 values were calculated as far as possible by Probit Analysis
Significant difference was tested with Dunnett-test

Any other information on results incl. tables

The test item had a statistically significant inhibitory effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after the exposure period of 72 hours at loading rates of 10 mg/L and above (results of Dunnett-tests, one-sided, a = 0.05, Tables 4 and 5). Thus, the loading rate of 10 mg/L was determined as the 72-hour LOEC (Lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined to be at the loading rate of 4.6 mg/L, since at this loading rate both, the mean biomass and the mean growth rates of the algae, were not statistically significantly lower than in the control.

The EC values were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration. All results are based on loading rates:

 

Parameter                                         Biomass b                   Growth rate r

(0-72 h)                                           (mg/L)                         (mg/L)

EC50                                               78                               > 100

95%-confidence limits                       n.d.                             n.d.

EC10                                               5.5                              9.1

95%-confidence limits                       n.d.                             n.d.

NOEC                                             4.6                              4.6

LOEC                                              10                               10

 

The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test media containing the test item at the loading rate of 22 mg/L and the algal cells in the controls. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including this loading rate.

In the control the cell density increased from nominal N = 1 x 104cells/mL at the start of the test (0 hours) to N = 48 X 104cells/mL (mean value) after 72 hours. Thus, the algal growth in the control was sufficiently high under the test conditions and the validity criterion of increase of cell density by at least a factor of 16 over the duration of the study was fulfilled.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

At the start of the test, the pH values in the test media and the control ranged from 7.9 to 8.0. At the end of the test, pH values between 8.0 and 8.9 were measured. The water temperature was constantly 22°C.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item ZP-TIX 1014 and its degradation products, respectively had a statistically significant inhibition effect on the growth (biomass and growth rate) of Scenedesmus subspicatus after 72 h at loading rate of 10 mg/L and above.
Since at the loading rate of 4.6 mg/L both the mean of biomass and the mean growth rate r of the algae were not satistically significantly lower than in the control, the value of 4.6mg/L was determined to be the NOEC; the highest concentration tested without toxic effect.
Executive summary:
The influence of the test item ZP-TIX 1014 on the growth of the green algal species Scenedesmus subspicatus CHODA T was investigated In a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3, 1992, and the OECD Guideline No. 201, 1984. The test method is also based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

In order to assess the toxicity of ZP-TIX 1014 to algae, five water accommodated fractions (WAFs) with loading rates of 4.6, 10, 22, 46 and 100 mg/L were tested as the test item contains components which may be sparingly soluble. Additionally, a control (test water without test item) was tested in parallel.

Five WAFs were prepared by dispersing adequate amounts of the test item in test water by stirring for 96 hours. After stirring, the dispersions of the test item were filtered through a membrane filter (pore size 0.45 µm). The filtrates of the dispersions were used as test media. Since the test item is not stable in test water, the degradation products were tested. Therefore, a long stirring period of 96 hours was chosen to enrich the test water with the maximum concentration of degradation products within a practically acceptable time period.

The test item is a poorly soluble mixture of isocyanates and it is generally known, that these compounds degrade fast in water. Due to these characteristics, the test item could not be analytically measured in the test media. Furthermore, the verification of the test item could not be based on its degradation products, since none could be defined during the analytical pre-experiments. For this reasons, no analytical work was performed.

The biological test results after 72 hours test duration:

The test item ZP-TIX 1014 and its degradation products, respectively, had a statistically significant inhibition effect on the growth (biomass and growth rate) of scenedesmus subspicatus after the exposure period of 72 hours at loading rates of 10 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects) and above. The 72-hour NOEC (highest concentration tested without toxic effects) was determined to be at the loading .rate of 4.6 mg/L, since at this loading rate both the mean biomass and the mean growth rater of the algae were not statistically significantly lower than in the control.

The EC values were calculated for both parameters, the algal biomass (b) and the growth rate (r), after 72 hours test duration. All results are based on loading rates:

 

Parameter                                         Biomass b                   Growth rate r

(0-72 h)                                           (mg/L)                         (mg/L)

EC50                                               78                               > 100

95%-confidence limits                       n.d.                             n.d.

EC10                                               5.5                              9.1

95%-confidence limits                       n.d.                             n.d.

NOEC                                             4.6                              4.6

LOEC                                              10                               10

 

n.d.: could not be determined