AAA reaction product

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 AAA reaction product in this semi-static test with daily test medium renewal, samples were taken from the freshly prepared test medium and the control at the start of the test (Day 0) and on the last day of test medium preparation (Day 3).

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 (Day 1) and at the end of the last test medium renewal period (Day 4).
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 in accordance with the guidelines to demonstrate that AAA reaction product has no toxic effect on the test fish 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 before the start of the test and prior to each test medium renewal by mixing 501 - 505 mg of AAA reaction product into 5000 mL test water using ultrasonic treatment for 15 minutes and intense stirring for 25 minutes at room temperature.

The test medium was freshly prepared just before introduction of the fish (= start of the test) and just prior to the test medium renewals.

The actual concentration of AAA reaction product (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 guidelines.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The study was performed with zebra fish (Brachydanio rerio). The test fish were obtained from Zoohaus Schaub, CH-4410 Liestal, Switzerland. In accordance with the test guidelines, the fish were held in the laboratories of RCC for more than two weeks without any medication. Prior to test start, they were acclimated for one week to the test water and temperature. During holding and acclimatization until one day before the start of the test the fish were fed with a commercial fish diet (Tetra Min Hauptfutter, supplied by TETRA-Werke, D-49304 Melle, Germany). During holding and acclimatization, the mortality rate in the batch of test fish was lower than 5% and all fish were healthy.

From the acclimated batch of test fish, 10 fish were measured at the start of the test: The mean body length of the fish was 3.2 ± 0.2 cm (mean ± standard deviation), the mean body wet weight was 0.27 ± 0.06 g.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

The test fish were observed after approximately 2½, 24, 48, 72, and 96 hours test duration for mortality and visible abnormalities.

Test conditions

Hardness:

250 mg CaCO3/L

Test temperature:

21-22°C

pH:

7.5-7.9

Dissolved oxygen:

8.3-8.7mg/L

Salinity:

pH=7.5-7.9

Nominal and measured concentrations:

Nominal: 100mg/L
Measured: 90-100% (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
One glass aquarium with 5 liters test medium was used for each treatment (the single test concentration and the control). The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification.

At the start of the test, 7 fish were introduced into each aquarium in a random order. The loading rate was 0.38 g fish wet weight per liter test medium. The test medium and the control were slightly aerated during the test period. The fish were not fed during the test.

A semi-static test with daily test medium renewal was performed to keep the test item concentration in the test medium as constant as possible during the test period of 96 hours. During this semi-static test, the test fish were transferred daily into a clean test vessel with freshly prepared test medium of the single concentration.


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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
A limit test was performed in accordance with the guidelines to demonstrate that AAA reaction product has no toxic effect on the test fish 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 before the start of the test and prior to each test medium renewal by mixing 501 - 505 mg of the test item into 5000 mL test water using ultrasonic treatment for 15 minutes and intense stirring for 25 minutes at room temperature.

The test medium was freshly prepared just before introduction of the fish (= start of the test) and just prior to the test medium renewals.

The actual concentration of AAA reaction product (i.e. of the different components of AAA reaction product) 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 guidelines.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The concentrations of AAA reaction product (diethanolamine, dimers, trimers, and polymers, and tetrahydrophthalic and trimellitic acids) were constant over the test medium renewal periods of 24 hours (90-100%). Therefore, the biological results were related to the nominal concentration of AAA reaction product.

In the control and in the test medium of 100 mg/L, all fish survived until the end of the test and no visible abnormalities were observed.

Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours) and the 96-hour LC0 of AAA reaction product to zebra fish were determined to be at least 100 mg/L. The 96-hour NOEC and the 96-hour LC0 might even be higher than this concentration but concentrations above 100 mg/L were not tested, according to the guidelines. The 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50, and the 96-hour LC100 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 tested concentration.

No remarkable observations were made concerning the appearance of the test medium. The test medium appeared to be a clear solution throughout the test medium renewal periods.

The pH values in the test medium and the control ranged from 7.5 to 7.9. The oxygen concentration was always 8.3 mg/L or higher, and thus higher than 60% oxygen saturation. The water temperature ranged from 21 to 22 °C.

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

NA

Any other information on results incl. tables

Sublethal observations / clinical signs:

The NOEC and the LC0 were determined directly from the raw data. The LOEC, the LC50, and the LC100 at the observation times could not be quantified due to the absence of a toxic effect of AAA reaction product at the tested concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of AAA reaction product to zebra fish (Brachydanio rerio) was determined in a 96-hour semi-static test in accordance to OECD 203. A limit test was performed up to and including the nominal concentration of 100 mg/L. The 96-hour NOEC and the 96-hour LC0 of AAA reaction product were determined to be at least 100 mg/L. The 96-hour LOEC, the 96-hour LC50 and the 96-hour LC100 were clearly higher than 100 mg/L.

Executive summary:

The acute toxicity of AAA reaction product to zebra fish (Brachydanio rerio) was determined in a 96-hour semi-static test in accordance to OECD 203. A limit test was performed up to and including the nominal concentration of 100 mg/L.

 

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 in the test medium of 100 mg/L, all fish survived until the end of the test and no visible abnormalities were observed during the test period of 96 hours. Hence, the 96-hour NOEC and the 96-hour LC0 of AAA reaction product were determined to be at least 100 mg/L. The 96-hour LOEC, the 96-hour LC50 and the 96-hour LC100 were clearly higher than 100 mg/L.