AAA reaction product

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-06-07 to 2004-11-19

Reliability:

1 (reliable without restriction)

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:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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:

The water temperature, pH-values, and oxygen concentrations were measured in the test medium and the control at the start of the test and once every day during the test in the freshly prepared and old test medium. At the same time, the appearance of the test medium was recorded.

For the determination of the actual concentration of the test item in the test medium, samples were taken from the single test concentration and the control just before the start of the test and after 48 hours. For the 48 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.

To confirm the maintenance of AAA reaction product concentration throughout the test medium renewal periods, samples were taken from the aged test medium and the control at the end of the first test medium renewal period and at the end of the last test medium renewal period.
Duplicate samples per sampling date were taken for each analysis, resulting in a total number of six samples per sampling date from the test medium and the control.

Test solutions

Vehicle:

no

Details on test solutions:

A limit test was performed to demonstrate that the test item has no toxic effect on Daphnia magna 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 by mixing 50.2 mg of the test item into 500 mL test water 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.

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.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with young daphnids of a clone of the species Daphnia magna Straus. A clone of this species was originally supplied by the University of Sheffield/UK in 1992, defined by the supplier as clone 5. Since that time, the clone has been bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests (see below).

At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Mobility of Daphnia Magna were evaluated after 24 hours and 48 hours

Test conditions

Hardness:

250 mg CaCO3/L

Test temperature:

20 - 21 °C

pH:

7.4 to 7.9

Dissolved oxygen:

At the beginning and the end of the test period, the dissolved oxygen concentrations in the test medium and the control were at least 8.6 mg/L.

Salinity:

pH=7.5-7.9

Nominal and measured concentrations:

Nominal: 100mg/L
Measured: 81-100% (diethanolamine, dimers, trimers, and polymers, and tetrahydrophthalic and trimellitic acids), hence constant over the test medium renewal periods of 24 hours.

Details on test conditions:

TEST SYSTEM
The test was performed in 100 mL glass beakers filled with 50 mL of test medium. The beakers were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions. The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification.

At the single test concentration and for the control, 20 daphnids were used divided into four replicates of five daphnids each. The daphnids were randomly distributed to the test vessels at initiation of the test. The loading rate was less than one daphnia per 5 mL of test solution.

A static test without test medium renewal was performed.

TEST MEDIUM / WATER PARAMETERS
Reconstituted water: analytical grade salts were dissolved in deionized water to obtain the following nominal concentrations:
CaCl2 x 2H2O : 2.0 mmol/L (= 294 mg/L)
MgSO4 x 7H2O: 0.5 mmol/L (= 123 mg/L)
NaHCO3 : 0.75 mmol/L (= 65 mg/L)
KCl : 0.075 mmol/L (= 5.8 mg/L)
Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO3)
Alkalinity : 0.8 mmol/L
Ratio of Ca : Mg = 4 : 1 (based on molarity)
Ratio of Na : K = 10 : 1 (based on molarity)

The test water was aerated prior to the preparation of the test medium until oxygen saturation was reached.

OTHER TEST CONDITIONS
- Adjustment of pH: No, pH=7.5-7.9
- 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 to demonstrate that AAA reaction product has no toxic effect on Daphnia magna 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 by mixing 50.2 mg of the test item into 500 mL test water 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 2.6.3).

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:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The concentrations of AAA reaction product (diethanolamine, dimers, trimers, and polymers, and tetrahydrophthalic and trimellitic acids) were constant over the test period (81-100%). Therefore, the biological results were related to the nominal concentration of AAA reaction product.

In the control and at the test concentration of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours.

Therefore, the 48-hour NOEC and the 48-hour EC0 of AAA reaction product to Daphnia magna were determined to be at least 100 mg/L. The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/L. These values could not be quantified due to the absence of toxicity of AAA reaction product at the test concentration of 100 mg/L.

The test medium appeared to be a clear solution throughout the test duration. At the start of the test, few fine particles were observed.

At the beginning and the end of the test period, the dissolved oxygen concentrations in the test medium and the control were at least 8.6 mg/L, the pH values were between 7.4 to 7.9 and the water temperature ranged from 20 to 21 °C.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control at least once a year. The latest result of the positive control test in 2004 (48 hour EC50: 0.83 mg/L, RCC Study no. 852355) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 1996 to 2004: 48-hour EC50: 0.55 - 1.1 mg/L).

Reported statistics and error estimates:

NA

Any other information on results incl. tables

The daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile).

 

The NOEC and EC0 were determined directly from the raw data.

 

The EC50 and the EC100 could not be determined due to the absence of a toxic effect of the test item in this test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of AAA reaction product to Daphnia magna was determined in a 48‑hour static test according to OECD 202. The 48-hour NOEC and the 48-hour EC0 of AAA reaction product to were determined to be at least 100 mg/L. The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/L. These values could not be quantified due to the absence of toxicity of AAA reaction product at the test concentration of 100 mg/L.

Executive summary:

The acute toxicity of AAA reaction product to Daphnia magna was determined in a 48‑hour static test according to OECD 202.

 

A limit test was performed to demonstrate that AAA reaction product has no toxic effect on Daphnia magna 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 AAA reaction product (diethanolamine, dimers, trimers, and polymers, and tetrahydrophthalic and trimellitic acids) in the test medium were quantified. The concentrations of diethanolamine, dimers, trimers, and polymers, and tetrahydrophthalic and trimellitic acids were constant over the test medium renewal periods of 24 hours, therefore, the biological results were related to the nominal concentration of AAA reaction product.

In the control and at concentration of 100 mg/L, no immobilized test organisms were observed during the test period of 48 hours.The 48-hour NOEC and the 48-hour EC0 of AAA reaction product to were determined to be at least 100 mg/L. The 48-hour EC50 and the 48-hour EC100 were clearly higher than 100 mg/L. These values could not be quantified due to the absence of toxicity of AAA reaction product at the test concentration of 100 mg/L.