AAA reaction product

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:

200406-04 to 2004-11-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study performed in accordance to guideline with no deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): DEA/ACID anhydride reaction product

- Physical state: highly viscous amber liquid

- Composition of test material, percentage of components:
Monomers: <17-35% (9-16% diethanolamine; 2-8% tetrahydrophthalic acid; 5-11% trimellitic acid; <0.1% tetrahydrophthalic anhydride; <0.8% trimellitic anhydride)
Dimers/trimers: <24-45%
Polymers: <10-25%
Not removable water: 5-15%

- Lot/batch No.:I=200554.UN2810; II=200501.UN2810. Batch no. 200554.UN2810 of AAA reaction product was used for the biological study. Batch no. 200501.UN2810 of AAA reaction product was additionally used for pre-experiments and analytical purposes.

- Expiration date of the lot/batch: I=October 2004; II==ctober 2004

- Stability under test conditions: stable under storage condition

- Storage condition of test material: in the refrigerator in the original container away from direct sunlight

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
NA

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Small volumes of the test medium and the control (1.0 mL) were taken out of all test flasks after 24, 48, and 72 hours of exposure, and were not replaced. The algal cell densities in the samples were determined by counting with an electronic particle counter (Coulter Counter, Model ZM), with at least two measurements per sample.

In addition, after 72 hours exposure, a sample was taken from the control and from the single test concentration. The shape of the algal cells was examined microscopically in these samples.

For the determination of the actual concentration of the AAA reaction product in the test medium, samples without algae were taken from the single test concentration and the control just before the start of the test and after 72 hours. For the 72 hour stability samples, additional flasks with adequate volumes of the freshly prepared test medium and the control were incubated under the same conditions as in the actual test but without algae.

Test solutions

Vehicle:

no

Details on test solutions:

A limit test was performed to demonstrate that AAA reaction product has no toxic effect on the algae up to and including the nominal test item concentration of 100 mg/L. Thus, a single test item concentration of nominal 100 mg/L was tested. Additionally, a control was tested in parallel (test water without addition of the test item).

The test medium was prepared immediately before the start of the test as follows: The amount of 60.3 mg of AAA reaction product was mixed into 600 mL test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature.

The actual concentration of AAA reaction product (i.e. of the different components of the test item) in the test medium was analytically determined (see Section 2.6.4).

The limit test was based on the results of a range-finding test and on the results of a pre-experiment to determine the solubility of AAA reaction product (without GLP). However, concentrations above nominal 100 mg/L were not tested according to the EU Commission Directive 92/69/EEC.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany). The algae had been grown in the RCC laboratories under standardized conditions according to the test guidelines.

Due to the division of the genus Scenedesmus into the genera Scenedesmus and Desmodesmus, Scenedesmus subspicatus was transferred to the new genus Desmodesmus. Thus Scenedesmus subspicatus was renamed as Desmodesmus subspicatus. However, the former name Scenedesmus subspicatus as specified in the guidelines is used in this report.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

Temperature controlled water bath at a temperature between 22 and 23 °C

pH:

7.7-8.4

Dissolved oxygen:

No information

Salinity:

No information

Nominal and measured concentrations:

Nominal: 100mg/L
Measured: 81-104% (diethanolamine, dimers, trimers, and polymers, and tetrahydrophthalic and trimellitic acids), hence constant over the test medium renewal periods of 72 hours.

Details on test conditions:

TEST SYSTEM
The algae were cultivated and tested in synthetic test water, prepared according to the test guidelines. The test was started (0 hours) by inoculation of 10,000 algal cells per mL test medium. The algal cells were taken from an exponentially growing pre-culture which was set up four days prior to the test under the same conditions as in the test. One day before the start of the test, the pre-culture was diluted threefold to keep the algae in exponential growth.

The test design included three replicates of the test concentration and six replicates of the control. Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erlenmeyer flasks.

The flasks were covered with glass dishes. They were incubated in a temperature controlled water bath at a temperature between 22 and 23 °C (Table 6) and continuously illuminated at a measured light intensity of about 8500 Lux (mean value), range: 8010 to 8910 Lux (minimum and maximum value of measurements at nine places distributed over the experimental area at the surface of the test media). This illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The test vessels were labeled with the RCC study number and all necessary additional information to assure unmistakable identification. The test duration was 72 hours.

TEST MEDIUM / WATER PARAMETERS
Analytical grade salts were dissolved in sterile purified water to obtain the following final nominal concentrations:

Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 x 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 x 7 H2O 15.0 mg/L
MgCl2 x 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L

Trace elements:
Na2EDTA x 2 H2O 100.0 µg/L
FeCl3 x 6 H2O 80.0 µg/L
MnCl2 x 4 H2O 415.0 µg/L
H3BO3 185.0 µg/L
Na2MoO4 x 2 H2O 7.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 x 6 H2O 1.5 µg/L
CuCl2 x 2 H2O 0.01 µg/L

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L as CaCO3).

OTHER TEST CONDITIONS
- Adjustment of pH: No, At the start of the test, the pH values in the test medium and the control were 7.7 and 8.1, respectively. At the end of the test, pH values of the test medium and the control were 8.2 and 8.4, respectively
- Photoperiod: A 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period
- Light intensity: ight intensity during the light period was approximately within the range of 50 to 500 Lux.

TEST CONCENTRATIONS
A limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that the test item has no toxic effect on the algae up to and including the nominal test item concentration of 100 mg/L. Thus, a single test item concentration of nominal 100 mg/L was tested. Additionally, a control was tested in parallel (test water without addition of the test item).

The test medium was prepared immediately before the start of the test as follows: The amount of 60.3 mg of the test item was mixed into 600 mL test water as homogeneously as possible using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature.

The actual concentration of the test item (i.e. of the different components of the test item) in the test medium was analytically determined (see Section 2.6.4).

The limit test was based on the results of a range-finding test and on the results of a pre-experiment to determine the solubility of the test item (without GLP). However, concentrations above nominal 100 mg/L were not tested according to the EU Commission Directive 92/69/EEC.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The mean algal cell densities in the treated test medium of 100 mg/L were nearly identical with or even slightly higher than those in the parallel control culture throughout the entire test duration. The mean biomass and the mean growth rate after 72 hours test duration were slightly higher than the control values.

Thus, AAA reaction product clearly had no inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours at the test concentration of 100 mg/L. This test concentration was therefore determined as the 72-hour NOEC. This value might even be higher but concentrations above 100 mg/L were not tested, according to EU Commission Directive 92/69/EEC.

The 72-hour LOEC and the 72-hour EC10 and EC50 for the algal biomass b and growth rate r could not be quantified due to the absence of toxic effect of AAA reaction product at the tested concentration. However, these values were clearly higher than 100 mg/L.

The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test medium containing the test item at a nominal concentration of 100 mg/L and the algal cells in the control. There were no obvious effects on the shape and size of the algal cells growing in the test medium.

In the control, the cell density increased from a nominal N = 1 x 104 cells/mL at the start of the test (0 hours) to N = 57 x 104 cells/mL (mean value) after 72 hours. Thus, the algal growth in the control was sufficiently high under the conditions of the test. Therefore, 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 medium. The test medium appeared to be a clear solution throughout the test duration.

At the start of the test, the pH values in the test medium and the control were 7.7 and 8.1, respectively. At the end of the test, pH values of the test medium and the control were 8.2 and 8.4, respectively. The water temperature ranged from 22 to 23 °C.

Results with reference substance (positive control):

For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control at least once a year to demonstrate satisfactory conditions of the test. The latest result of the positive control test in 2003 (72-hour EC50 for the growth rate: 0.69 mg/L, RCC Study 849621) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2003: 72-hour EC50: 0.44 - 1.16 mg/L).

Reported statistics and error estimates:

NA

Any other information on results incl. tables

AUC (area under the curve) and r (growth rates) calculated for each test flask. Based on these values, the arithmetic mean area and the arithmetic mean growth rate were calculated for all test flasks per treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.

 

A statistical evaluation of the inhibition of biomass and growth rate was not performed since the mean biomass and the mean growth rate after 72 hours test duration were even slightly higher than the control values.

 

The test concentrations corresponding to 10 and 50% inhibition of the algal biomass b (E_bC10, E_bC50), or of the growth rate r (E_rC10, E_rC50) could not be calculated since a limit test was performed.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The influence of AAA reaction product on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test in accordance to OECD 201. AAA reaction product had clearly no inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours at the test concentration of 100 mg/L. The 72-hour NOEC of AAA reaction product to Scenedesmus subspicatus was determined to be at least 100 mg/L. The 72-hour LOEC and the 72-hour EC10 and EC50 for the algal biomass b and growth rate r could not be quantified due to the absence of a toxic effect of AAA product at the tested concentration. However, these parameters were clearly higher than 100 mg/L.

Executive summary:

The influence of AAA reaction product on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test in accordance to OECD 201.

 

A limit test was performed to demonstrate that AAA reaction product has no toxic effect up to and including the nominal concentration of 100 mg/L. Thus, the only concentration tested was nominal 100 mg/L, alongside a control.

 

Different components of the test item (diethanolamine, dimers, trimers, and polymers, and tetrahydrophthalic and trimellitic acids) in the test medium were quantified and were shown to be constant over the test period of 72 hours. Therefore, the biological results were related to the nominal concentration of AAA reaction product.

AAA reaction product had clearly no inhibitory effect on the growth of Scenedesmus subspicatus during the exposure period of 72 hours at the test concentration of 100 mg/L. The 72-hour NOEC of AAA reaction product to Scenedesmus subspicatus was determined to be at least 100 mg/L. The 72-hour LOEC and the 72-hour EC10 and EC50 for the algal biomass b and growth rate r could not be quantified due to the absence of a toxic effect of AAA product at the tested concentration. However, these parameters were clearly higher than 100 mg/L.