2,20:3,19-Dimethano-2,3,4a,5a,6a,7a,8a,9a,10a,11a, 12a,13a,14a,15a,16a,17a,19 ,20,21a,22a,23a,24a,25a,26a,27a,28a,29a,30a,31a,32a,33a,34a-dotriacontaazabispentaleno [1''',6''':5'',6'',7''] cycloocta[1'',2'',3'':3'',4'']pentaleno[1'',6'':5',6',7']cycloocta[1',2',3':3',4']pentaleno[1',6':5,6,7]cycloocta[1,2,3-gh:1',2',3'-g'h']cycloocta[1,2,3-cd:5,6,7-c'd']dipentalene-1,4,6,8,10,12,14,16,18,21,23,25,27,29,31,33-hexadecone, hexadecahydro-, stereoisomer,2,18:3,17-Dimethano-2,3, ,4a,5a,6a,7a,8a,9a,10a,11a,12a,13a,14a,15a,17,18,19a, 20a,21a,22a,23a,24a, 25a,26a,27a,28a,29a,30a-octacosaazabispentaleno[1''',6''':5'',6'',7'']cycloocta [1'',2'',3'':3'',4''] pentaleno[1'',6'':5',6',7']cycloocta[1',2',3':3',4']pentaleno[1',6':5,6,7]cycloocta[1,2,3-cd:1',2',3'-gh]pentalene-1,4,6,8,10,12,14,16,19,21,23,25,27,29-tetradecone, tetradecahydro-, stereoisomer, 1H,4H,12H,15H-2,14:3,13-Dimethano-5H,6H,7H,8H,9H,10H,11H,16H,17H,18H,19H,20H,21H,22H-2,3,4a,5a,6a,7a,8a,9a,10a,11a,13,14,15a,16a,17a,18a,19a,20a,21a,22a-eicosaazabispentaleno [1'',6'':5',6',7']cycloocta[1',2',3':3',4']pentaleno[1',6':5,6,7]cycloocta[1,2,3-cd:1',2',3'-gh]pentalene-1,4,6,8,10,12,15,17,19,21-decone, decahydro-, stereoisomer

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:

19 July 2018 to 14 November 2018 (experimental completion date)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analysis of the test dispersions was carried out by TOC analysis. Water samples were taken from the control and test group at 0, 24, 48 and 72 hours from fresh media and at 24, 48, 72 and 96 hours from old media for TOC analysis. All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

For the range-finding test, a nominal amount of test item (4000 mg) was dispersed in test water and the volume adjusted to one liter to give a 4000 mg/L stock solution from which a series of dilutions was made to give the test concentrations of 1.0, 10 and 100 mg/L.
To ensure adequate mixing and homogeneity, the stock solution was inverted several times and each of the test preparations were mixed with a flat bladed stirrer for approximately one minute.

For the definitive test, a nominal amount of test item (2000 mg) was dispersed in test water and the volume adjusted to one liter to give a 2000 mg/L stock solution. The stock solution was inverted several times to ensure adequate mixing and homogeneity.
A dilution was made from the stock solution to give 20 litres of media at the test concentration of 100 mg/L. The test preparation was mixed with a flat bladed stirrer for approximately one minute.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 14 August 2018 to 21 August 2018. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The stock fish were fed commercial trout pellets which was discontinued approximately 27 hours prior to the start of the definitive test. There were two mortalities in the 7 days prior to the start of the test. The fish had a mean standard length of 4.7 cm (standard deviation = 0.4) and a mean body weight of 0.77 g (standard deviation = 0.28) at the end of the definitive test. Based on the mean body weight value this gave a loading rate of 0.27 g body weight/liter.

Test type:

semi-static

Water media type:

freshwater

Remarks:

The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish. Laboratory tap water was dechlorinated and partly softened.

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Approximately 140 mg/L as CaCO3.

Test temperature:

The water temperature was controlled at 14 °C to 15 °C.

pH:

A 100 mg/L solution of the test item was made up in de-chlorinated tap water and the pH was measured at 7.8. Therefore, no pH adjustment was required during testing.

Dissolved oxygen:

The dissolved oxygen content was greater than or equal to 8.9 mg O2/L.

Nominal and measured concentrations:

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 72 hours showed that measured concentrations of between 20 and 24 mg C/L were achieved. Analysis of the test preparations at 24 and 96 hours showed that measured concentrations of between 28 and 27 mg C/L were achieved.

Details on test conditions:

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
In the range finding test three fish were placed in each test and control vessel.
Each 25 to 30 liter test and control vessel contained 20 liters of test media and was covered to reduce evaporation. After 1, 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.
The control group was maintained under identical conditions but not exposed to the test item.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The test concentration to be used in the definitive test was determined by a preliminary range finding test. In the range finding test fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/L. The test item was dissolved directly in test water. There were no sub-lethal effects of exposure during the range finding test at the test concentrations of 1.0 and 10 mg/L. However, fish sitting on the bottom of the vessel were observed at 100 mg/L. No sub-lethal effects were observed in the definitive test.

Validity criteria fulfilled:

yes

Conclusions:

There were no mortalities in seven fish exposed to a test concentration of 100 mg/L for a period of 96 hours. The LC50 is > 100 mg/L There were no sub-lethal effects of exposure observed in the test

Executive summary:

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Following a preliminary range‑finding test, seven fish were exposed to an aqueous dispersion of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of 14 ºC to 15 °C under semi‑static test conditions. The number of mortalities and any sub‑lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀ values based on the nominal test concentration of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Description of key information

The NOEC in rainbow trouts was at 100 mg/L, the highest tested concentration in a GLP study performed in accordance with current OECD and EU test guidelines.