7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Feb 2000 - 16 Jul 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Difficulties in appropriate preparation of the test solution and recovery of the test item in measurements.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: test solutions prepared for renewals and the concentrations of 24 h old medium (directly before renewal) were analyzed every 24 h

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Due to difficulties of the appropriate preparation of the test solution and subsequent interpretation of the study results and in order to get information on whether the test substance concentration or unsolved particles/ drops were responsible for the clear effect, limit tests were conducted with solutions prepared in three different ways, each including an untreated control vessel.
- Method:
1). Test A:
The pure test substance was weighed into the vessels directly to prepare a stock solution for every test solution. Stock solutions were shaken for 24 h and then diluted with purified drinking water to the test concentrations. Purified drinking water was tested as control. The results are referred to as test A.
2). Test B:
The bad solubility of the test substance led to difficulties of interpretation of the study results. In order to get information on whether the test substance concentration or unsolved particles/ drops were responsible for the clear effect, the study protocol was added by limit tests with nominal concentrations between 1000 mg/L and 333 mg/L, which were prepared in three different ways, each including an untreated control vessel. The results are referred to as tests B1, B2, and B3.

B1) An eluate of the test substance was prepared by giving 15 g of the test substance to 1 L water followed by stirring for 24 h. The eluate was filtered by using a glass fiber filter. 2/3 of the eluate were diluted in 10 L test solution (nominal concentration of 1000 mg/L), as well as the remaining 1/3 (nominal concentration of 500 mg/L).

B2) Eluates of the test substance were prepared by giving two times 5000 mg of the test substance to 5 L of water each followed by stirring for 24 h. The eluates were filtered by using a glass fiber filter and combined to the test solution of 10 L (nominal concentration of 1000 mg/L). At the same time the procedure was repeated with two times 2500 mg (nominal concentration of 500 mg/L).

B3) Eluates of the test substance were prepared by giving two times 5000 mg of the test substance to 5 L of water each followed by stirring for 24 h. The eluates were filtered by using a glass fiber filter and diluted to the test solution of 10 L by 1:2 (nominal concentration of 500 mg/L) and 1:3 (nominal concentration of 333 mg/L).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: laboratory bred, originally obtained from est Aquarium GmbH, Bad Lauterberg, Germany
- Length at study initiation: 2.6 cm (mean of 30 fish)
- Weight at study initiation: 0.26 g (mean of 30 fish)
- Method of breeding: fish were acclimatized in the same water as used in the test. They were fed daily with TetraMinR Hauptfutter
- Feeding during test: no feeding occurred during the test as well as 24 h before test start

ACCLIMATION
- Acclimation conditions: fish were acclimatized in water of the same quality as used in the test
- Type and amount of food: TetraMin Hauptfutter (Tetra Werke, Melle, Germany)
- Feeding frequency: fed daily
- Health during acclimation (any mortality observed): only healthy fish without diseases and abnormalities were used in the study

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21.9 - 22.1 °C (given for test A as explained in field "details on test solutions")

pH:

7.7 - 8.0 (given for test A as explained in field "details on test solutions")

Dissolved oxygen:

7.4 - 8.7 mg/L (given for test A as explained in field "details on test solutions")

Nominal and measured concentrations:

0 (control), 95.3, 171.5, 308.6, 555.6 and 1000.0 mg/L (nominal)
0, 6.5, 7.2, 8, 10.7, 12.4 mg/L (mean measured as determined in test A)

Details on test conditions:

TEST SYSTEM
- Test vessel: 15 L glass aquaria
- Fill volume: 10 L
- Aeration: solutions were aerated via a glass capillary
- Renewal rate of test solution: every 24 h
- No. of organisms per vessel: 10 fish
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: test A: 0.3 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified drinking water purification through filtration with charcoal, aerated and passed through a lime stone column. Water was aerated to oxygen saturation prior to preparation of the test solutions
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH, oxygen concentration and temperature were measured directly before adding the fish and afterwards once per day

OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle was adjusted to 14/10 h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and abnormalities were recorded after 24, 48, 72 and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

10.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI: 9.6 – 10.7 mg/L

Remarks:

Results refer to test A

Details on results:

- Other: effect concentrations refer to the mean measured concentrations, as the measured concentrations deviated by more than 20% from the nominal values

Reported statistics and error estimates:

The LC50 and LC10 values as well as the confidence limits of the LC50 were derived by probit analysis.

Sublethal observations / clinical signs:

Results Test A:

Table 1: Test concentrations (mg/L) - nominal and measured concentrations of the test substance.

Nominal test concentrations	Measured test concentrations						Deviations (%) from nom. values
	0 h	24 h	* 48 h	* 72 h	* 96 h	Mean ± s.d.
Control	0	0.0 / 0.0	0.5 / 0.0	0.2 / 0.0	0	0.1 ± 0.2
95.3	3.54	4.1 / 3.6	7.2 / 12.1	9.8 / 3.8	7.5	6.5 ± 3.3	-93.2
171.5	3.47	6.4 / 4.0	8.4 / 7.9	11.5 / 5.5	10.2	7.2 ± 2.8	-95.8
308.6	3.75	7.0 / 5.0	9.9 / 8.8	13.3 / 5.6	10.4	8.0 ± 3.2	-97.4
555.6	4.3	7.6 / 13.0	16.3 / 9.5	14.7 / 6.4	13.4	10.7 ± 4.3	-98.1
1000	6.15	8.8 / 13.9	17.1 / 12.3	15.9 / n.d.	n.d.	12.4 ± 4.2	-98.8

* test concentration before renewal of the test solution / freshly prepared test solution.

Table 2: Cumulative mortality during the test period of 96 h.

Measured test concentrations (mg/L)		Cumulative mortality
Mean	Maximum	24 h	48 h	72 h	96 h
Control	0.5	0	0	0	0
6.5	12.1	0	0	0	0
7.2	11.5	0	0	0	0
8	13.3	0	0	0	0
10.7	16.3	0	6	7	7
12.4	17.1	0	9	10	10

Table 3: Further lethal effect concentrations (mg/L) of the test substance.

Hours of test	NOEC	LOEC	LC10
96	8	10.7	9.4

Results Test B:

B1:

In test B1 the two vessels were measured with concentrations clearly below the NOEC of test A (mean values: 0.8 and 2 mg/L) and showed no mortality until 48 h when the test was terminated.

B2:

In test B2 the two vessels were measured with concentrations above the LC50 of test A (mean values: 19 and 25 mg/l) and showed 100% mortality after 24 h.

B3:

Table 4: Measured concentrations and cumulative mortality during the test B3

Test concentrations (mg/L)			Cumulative mortality
Nominal	Measured
	Mean	Maximum	24 h	48 h	72 h	96 h
Control	0	0	0	0	0	0
333	10.7	11.5	0	0	2	2
500	12.8	17.2	3	4	5	6

Other parameters measured in Test B3: temperature: 21.8 - 22.0 °C, dissolved oxygen: 7.6 - 8.9 mg/L, pH: 7.7 - 8.4, biomass loading rate: 0.2 mg/L.

LOEC of test A was determined with 10.7 mg/L (related to mean concentrations), and exactly met by the mean value of the lower concentration of test B3, at which 20% of the fish died. Mortalities of 70 to 100% occurred in test A at mean concentrations of 10.7 and 12.4 mg/L, compared to 60% mortality at 12.8 mg/L in test B. The data of tests B strongly support the effect concentrations derived from test A and the conclusion that no particles or drops were responsible for the measured effect.

Validity criteria fulfilled:

yes

Description of key information

LC50 (96 h) = 10.1 mg/L (measured, geom. mean) for Danio rerio (OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

10.1 mg/L

Additional information

One study is available, in which the short term toxicity of the substance (CAS 72869-86-4) to fish was investigated. The study was assessed according to GLP and OECD guideline 203. Due to difficulties of the appropriate preparation of the test solution and in order to get information on whether the test substance concentration or unsolved particles/ drops were responsible for effect, tests were conducted with solutions prepared in different ways. In the main test (Test A), pure test substance was weighed into the vessels directly to prepare a stock solution for every test solution. Stock solutions were shaken for 24 h and then diluted with purified drinking water to the nominal test concentrations of 95.3, 171.5, 308.6, 555.6 and 1000.0 mg/L. In a semi-static test system the test organism Danio rerio, was exposed to these nominal test concentrations for 96 h. An analytical monitoring was performed via HPLC-UV. The recoveries of the test substance were very low and mean measured concentrations ranged between 6.5 and 12.4 mg/L (geom. mean). Lethal effects were observed at the two highest concentrations resulting to an EC50 (96 h) of 10.1 mg/L (measured, geom. mean).