Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Because of the poor solubility of the test item, no calculation from content of organic carbon of the test item and DOC measurement was performed.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The water-accommodated fractions were prepared for the test. This was done mixing the nominal loads with the corresponding amount of nutrient medium and shaking vigorously for 24 hours. The resulting solutions were filtrated through 0.45 µm filters.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga (Desmodesmus subspicatus)
- Strain: CHODAT
- Source, Method of cultivation: The culture of Desmodesmus subspicatus was obtained in Oct. 2011 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
- Age of inoculum (at test initiation):
Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into pre-culture medium and incubated for 96 hours. The resulting culture is growing exponentially.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not reported.

Test temperature:

24 °C

pH:

7.7 - 8.6

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Nominal and measured concentrations:

4.6 / 10 / 22 / 46 / 100 mg/L nominal concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks, total volume 50 mL
- Aeration: No
- Initial cells density: 2 *10^3 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Not reported
- Light intensity and quality: 5800 Lux
- Salinity (for marine algae): n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: calculated based on the determination of the absorption at 440 nm
- At the end of the test, the treatments were examined microscopically in order to assess the appearance of the alga and detect abnormalities (e.g. caused by the exposure to the test item).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

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:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: None
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.1 mg/L
- Other: NOErC = 0.16 mg/L

Reported statistics and error estimates:

t-test
F-test

Growth Rate µ, Area under the Curve AUC, Yield Main Study

NominalConcentrationin mg/L		Growth Rate [day^-1]	AUC [Cell Concentration/mL*day]	Yield [Cell Concentration/mL]
0	Mean	1.43	138978	159200
	SD	0.05	15556	20819
4.6	Mean	1.39	119859	143390
	SD	0.06	18244	25797
10	Mean	1.35	114161	125374
	SD	0.05	17123	19895
22	Mean	1.48	158097	182363
	SD	0.01	4176	7091
46	Mean	1.39	125374	141919
	SD	0.09	28242	36488
100	Mean	1.56	238616	240454
	SD	0.05	41094	36164

SD = Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

In a GLP-study according to OECD Test Guideline 201, the registered substance was not toxic to algae up to a nominal concentration of 100 mg/L.

Executive summary:

A study according to OECD 201 (resp. EU C.3) was performed to evaluate the toxicity of the registered substance to the alga Desmodesmus subspicatus.

The study was performed using five concentrations ranging from 4.6 to 100 mg/L nominal concentration. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the adsorption and the cell density of the cultures determined by microscope counts. Growth rate µ, area under the growth curve (AUC) and the yield were determined from the cell densities at the respective observation times.

Significant inhibition of algal growth was not observed in any of the treatments compared to the control.

At the beginning and at the end of the test, the content of the test item in the test solutions was determined using DOC-determination. Because of the very low solubility in test medium, no calculation from content of carbon of the test item and DOC measurement was possible. The measured start concentration in the treatments was only slightly above the LOQ. At the end of the test, the concentration of DOC in the treatments and in the control was in the same range. That means that in presence of the test system, the DOC was affected by the algae.

Therefore, the determination of the biological results was based on the nominal loadings.

The EC50s of potassium dichromate were tested in a separate reference test (GLP study no. 201206R301). The values lay within the normal range of the laboratory.

The following results for the test item CARDOLITE NC-513 were determined:

Endpoint	NOEL	LOEL	EL10	EL50
Growth Rate	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L
AUC^1	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L
Yield ^2	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L

1 AUC (Area Under Curve according to EU Method C.3) means the integral of the biomass. Calculation see under 8.3.

2 Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period. Calculation see under 8.4

Description of key information

- EL50 (72h) for freshwater algae (Pseudokirchneriella subcapitata): >100 mg/l [nominal; based on both growth rate and yield; OECD 201]

- NOEL (72h) for freshwater algae (Pseudokirchneriella subcapitata): ≥100 mg/l [nominal; based on both growth rate and yield; OECD 201]

- LOEL (72h) for freshwater algae (Pseudokirchneriella subcapitata): >100 mg/l [nominal; based on both growth rate and yield; OECD 201]

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

In a GLP-study according to OECD Test Guideline 201, the registered substance was not toxic to algae up to a nominal concentration of 100 mg/L.