A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to a current approved guideline and following good laboratory practices.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

87787-81-3

EC Number:

618-070-1

Cas Number:

87787-81-3

IUPAC Name:

87787-81-3

Constituent 2

Reference substance name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

EC Number:

413-800-3

EC Name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Cas Number:

87787-81-3

Molecular formula:

C44H79NO3

IUPAC Name:

2-(dioctadecylcarbamoyl)benzoic acid; 2-(ditetradecylcarbamoyl)benzoic acid; 2-[(propan-2-yloxy)carbonyl]benzoic acid; 2-[octadecyl(tetradecyl)carbamoyl]benzoic acid; 2-octadecyl-2,3-dihydro-1H-isoindole-1,3-dione; 2-tetradecyl-2,3-dihydro-1H-isoindole-1,3-dione; benzene-1,2-dicarboxylic acid; dioctadecylamine; ditetradecylamine; octadecyl(tetradecyl)amine

Constituent 3

Reference substance name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

IUPAC Name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

Constituent 4

Reference substance name:

Stepan TAB-2

IUPAC Name:

Stepan TAB-2

Details on test material:

Test substance was treated as 100% pure. Test substance is stable at 50 deg C and Stored at room temperature in the dark. Expiry date April 30, 1992. Test substance batch number: 198308.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samping for analysis of test concentrations: During the final test duplicate samples from 0, 1000 mg/l (Vessel B and C) and Tween-control were taken for analysis. During the test also homogeneity was tested in vessel A: i.e. duplicate samples were taken at t=0 and t=24 hours, from just below the surface of the medium, from the middle and from just above the bottom of the test vessel.
Sampling Frequency: At t=O hours (fresh medium) and t=24 hours (old medium before renewal)
Sampling volume: 10 ml from the approximate center of the test vessels
Storage: Not applicable, samples were analyzed immediately.

Test solutions

Vehicle:

yes

Details on test solutions:

Test vessels: 5 liters, all glass.
Frequency of renewal of medium: every 24 hours
Test Medium: Dutch standard water (DSW) formulated using Milli-Ro water according to Adema (Adema D.M.M., 1980; In: Degradability, Eco-toxicity and Bioaccumulation: The determination of the possible effects of chemicals and wastes on the aquatic environment, Part II, Chapter 5, Government Publishing Office, The Hague, The Netherlands).
Dutch Standard Water (DSW) had the following composition:
Ca+2 54.4 mg/l
Mg+2 17.5 mg/l
Na+ 7.8 mg/l
Cl- 96.6 mg/l
SO4-2 70.1 mg/l
Co3-2 --1)
HCO3- 84.8 mg/l 1)
After aeration the hardness was 210 mg/CaCO3 per liter and the pH was 8.2± 0.2
Milli-Ro water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA)

Test organisms

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species: Carp (cyprinus carpio, Teleostei, Cyprinidae) (Linnaeus, 1758)
Source: Zodiac, proefacc, "De Haar Vissen," L.U. Wageningen, The Netherlands
Mean length: 2.0 +- 0.011 cm
Mean weight: 0.12 +- 0.018 g
Characteristics: Pathogen-free F1 from a single parent-pair
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 80
Acclimation period: At least 14 days after delivery
Feeding: Daily with Trouvit or Artemia
Validity of batch: Following a 48-hour settling in period the fish were allowed to acclimatise to the test medium witout test substance for at least seven days. In the batch of fish used for the test mortality during the seven days prior to the start of the test was less than 5%.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No data provided.

Test conditions

Hardness:

210 mg CaCO3 (Note: value might have changed during the test as a result of continuous aeration of the Dutch Standard water)

Test temperature:

The temperature of the test medium measured in the blank control ranged from 21.5 to 22.5°C.

pH:

The pH of the fresh media of the treated groups ranged from 6.4 to 6.8. During the 24 hours before renewal pH increased 0.9 to 1.5 units, i. e., varying from 7.6 to 8.0. The pH in the controls ranged from 8.0 to 8.4.

Dissolved oxygen:

> 5 mg/l for all measurements performed during the final study.

Salinity:

No data provided.

Nominal and measured concentrations:

Limit test with 1000 mg/l nominal concentration, which is the maximum concentration to be tested. The number of fish exposed to test substance was 30, with duplicate runs. The number of controls were 10 fish per run.

Details on test conditions:

The test type was semi-static for 96 hours.
Test vessels: 5 liters, all glass.
Frequency of renewal of medium: every 24 hours
Test Medium: Dutch standard water (DSW) formulated using Milli-Ro water according to Adema (Adema D.M.M., 1980; In: Degradability, Eco-toxicity and Bioaccumulation: The determination of the possible effects of chemicals and wastes on the aquatic environment, Part II, Chapter 5, Government Publishing Office, The Hague, The Netherlands).
Dutch Standard Water (DSW) had the following composition:
Ca+2 54.4 mg/l
Mg+2 17.5 mg/l
Na+ 7.8 mg/l
Cl- 96.6 mg/l
SO4-2 70.1 mg/l
Co3-2 --1)
HCO3- 84.8 mg/l 1)
After aeration the hardness was 210 mg/CaCO3 per liter and the pH was 8.2± 0.2
Milli-Ro water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA)
1) It should be noted that the DSW was aerated continuously before and during use in the tests which might have changed these values.

Controls: Test medium without test substance or other additives (0 mg/l) and one control containing test medium without test substance but with the additive used in the treatment of the stock solutions, i.e. Tween 80 (0.1 ml/l).
Number of fish: 30 fish/ per test concentration per run
Controls: 10 fish per run
Loading: 0.4 g fish/liter, i.e., 10 fish per 3 liters of test medium
Illumination: 16 hours photoperiod per day
Aeration: The test media were aerated continuously during the final test.
Feeding: No feeding from 24 hours prior to the test and during the total test period.
Introduction of fish: Directly after preparation of the test media fish were introduced into the test medium, provided that the temperature of the medium was within the optimal range.
Preparation of the test media: Daily a weighed amount of test substance (2908 ± 133 mg) was melted at 50°C. Then it was mixed with Tween 80 (0.3 ml). This mixture was added quantitatively to 3 liters of DSW (50°C) providing a nominal concentration of 1000 mg/l. Ultra sonication was applied to ca. 22°C under continuous aeration. The final test solutions were turbid.
Sampling for analysis of test concentrations: During the final test duplicate samples from 0, 1000 mg/l (Vessel B and C) and Tween-control were taken for analysis. During the test also homogeneity was tested in vessel A: i.e. duplicate samples were taken at t=0 and t=24 hours, from just below the surface of the medium, from the middle and from just above the bottom of the test vessel.
Sampling Frequency: At t=O hours (fresh medium) and t=24 hours (old medium before renewal)

Sampling volume: 10 ml from the approximate center of the test vessels
Storage: Not applicable, samples were analyzed immediately.
Additionally, reserve samples of 25 ml were taken from all groups. These samples will be stored at -20°C for an additional three months after delivery of the drat report, pending on the decision of the sponsor for additional analysis. The method of analysis is described in the appended Analytical Report.
Measurements and Recordings:
Mortality and other effects: At 3, 24, 48, and 96 hours following the start of exposure. Dead fish were removed when observed.
Fish length and weight: Ten fish of the batch used for the test were weighted and measured prior to the start of the test.
Dissolved oxygen content and pH: Measured and recorded daily in all vessels, beginning at the start of the test.
Temperature of medium: Measured daily in one control vessel, beginning at the start of the test.
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to about 1.2% ethylene glycol monophenyl ether in water.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Nominal concentration corresponds with an actual concentration ranging from 78 to 337 mg/l defined as the sum of dissolved and undissolved (dispersed) test substance particles.

Details on results:

The results of analysis of the samples taken during the study are described in the appended analytical report. The actual concentration was based on analysis of both dissolved and undissolved test substance particles.

Analysis of the samples taken at the start (t=0) of the final test revealed that only part of the 1000 mg/l was present in the water phase, i.e., actual concentrations ranging from 130 to 222 mg/l. Similar results were found after 24 hours before renewal. In some cases the variation between the values measured in the duplicate samples was relatively high. During the test a part of the undissolved test substance particles was floating on the surface and another part was present as deposit. These observations confirm the relatively low recovery found in the water phase.

Analysis of the samples taken for the homogeneity testing revealed that Stepan TAB-2 was not completely homogeneously dispersed through the test medium. The values measured ranged from 78 mg/l (sample from the upper layer) to 337 mg/l (sample from the bottom layer). After 24 hours the concentrations measured in both the upper (90% height) and bottom layer (10% height) of the dispersion were significantly lower than the initial ones; those in the sample from the center (50% height) were in the same range as the initial ones. This indicated that the dispersion was not stable, and the decrease observed was probably due to the deposition of test sample particles.

Results with reference substance (positive control):

This reference test was carried out to check the sensitivity of the test system as used by RCC NOTOX. The positive control substance was pentahlorophenol (PCP, SIGMA, Art. P8519, Batch 113F-3504) was obtained from Brunschwig Chemie, Amsterdam, The Netherlands. Concentrations employed wire: 0.1, 0.18, 0.32, 0.56, and 1.9 mg/l in DSW. Control was DSW (Dutch Standard Water) without test substance.

During the test ;H, dissolved oxygen content and the temperature of the medium were within the optimal ranges for fish. The 96 hour LC50 of this test is 0.33 mg/l with a 95% confidence interval between 0.28 and 0.47 mg/l. The historical range of the 96 hour LC50 for carp recorded in reference tests approximately every 3 months since April 1988 is between 0.18 and 1.0 mg/l.

Reported statistics and error estimates:

No data provided.

Any other information on results incl. tables

Sublethal observations / clinical signs:

No LC50 could be calculated because the test substance proved to be non-toxic (LC50 > maximum concentration).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Acceptability of the test: The mortality in the blank and, if relevant, in the control containing the vehicle should not exceed 10%., This was what was observed.

Conclusions:

The 96-hour LC50 for carp exposed to an unstable dispersion of Stepan TAB-2 in water was greater than nominally 1000 mg/l, corresponding with an actual concentration ranging from 78 to 337 mg/l defined as the sum of dissolved and undissolved (dispersed) test substance particles.

Executive summary:

96 Hour Acute Toxicity Study in the Carp with Stepan TAB-2.

The final study was a limit test because no toxicity was observed in the range-finding test. In this limit test 2 x 10 fish were exposed for 96 hours in a semi-static system to the nominal concentrations of 1000 mg/l. The test solutions were turbid dispersions with undissolved test substance particles. All solutions were renewed daily.

During 96 hours of exposure no mortality of fish or any other effects were observed at the nominal concentration of 1000 mg/l.

The 96 -hour LC50 for carp exposed to Stepan TAB-2 was greater than nominally 1000 mg/l, corresponding with an actual concentration ranging from 78 to 337 mg/l defined as the sum of dissolved and undissolved (dispersed) test substance particles.