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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-02-2002 / 26-09-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study peformed according to standard protocol and under GLP. Validity criteria can be considered as fulfilled, immobilisation of control animals was <10%, the control animals were not staying at the surface, the concentration of dissolved oxygen in all test vessels was >3 mg/L and the concentration of test substance >= 80% of initial concentration during test. Although quantification of the applied concentrations was not possible due to the absence of a suitable analytical method. Therefore only the stock solution used to prepare the test concentrations were analyzed. These analyses showed that the stock solution was stable even after storage for almost 7 months in the refrigerator. Considering the high water solubility of the test substance and its stability it is reasonable to assume that the actual concentrations were identical to the nominal concentrations during the entire test. A dose-response related increase in mortality was observed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test substance was soluble in water. A stock solution of approximately 0.1 g/l of test substance was prepared as follows: to an accurately measured amount of 0.1127 g of test substance approximately 120 ml of deionized water of approximately 50C was added. The test substance dissolved well under stirring. After cooling of deionized water was added up to a final volume of 1000 ml. A homogeneous, clear stock solution was obtained. It was kept stirring at room temperature before being used for the preparation of the test solutions at the start of the test.
The test solutions were prepared by addition of the required amounts of stock solution to the test medium to obtain the following concentrations: 0.01 – 0.02 – 0.04 – 0.08 and 0.16 mg/l. A control containing only test medium was included in the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was performed with Daphnia magna (water fleas), which where obtained from Aquasense at Amsterdam. The animals used in the test were less than 24 hours old at the beginning of the test and were obtained from parent animals having an age of 2-4 weeks.
Test type:
static
Limit test:
no
Test temperature:
The temperature in the test vessels during the test period ranged from 20.6 to 21.4 °C.
pH:
The maximum variation of pH observed during the test was 0.2 units.
Dissolved oxygen:
The maximum variation in oxygen concentration during the test was 0.3 mg/L
Salinity:
As test medium so called Dutch Standard water was used, with a pH of approximately 8.2 and a hardness of approximately 12°dH, containing per liter of deionized water: 100 mg of NaHCO3, 20 mg of KHCO3, 200 mg of CaCl2•2H2O and 180 mg of MgSO4•7H2O. The test medium was aerated before being used in the test. The air was water-saturated and purified by an active coal and cotton filter.
Nominal and measured concentrations:
The available analytical method was not suitable to quantify concentrations below 10 mg/L. Therefore only the stock solutions used to prepare the test concentrations were analyzed. The measured concentration of the undiluted stock solution (109.8 mg/L) differs only slightly from the nominal concentration of the stock solution (112.7 mg/L). These results show that the stock solution was stable even after storage for almost 7 months in the refrigerator. All concentrations mentioned in this report are therefore based on nominal concentrations. Considering the high water solubility of the test substance and its stability it is reasonable to assume that the actual concentrations were identical to the nominal concentrations during the entire test.
Details on test conditions:
The test was performed as a static test. The total duration was 48 hours. 20 daphnids divided into 4 batches of 5 animals were used per test concentration and control.

Under otherwise identical test conditions, the daphnids were exposed to the chosen concentrations of the test substance as described below and immobility and sub-lethal effects were recorded at approximately 24 and 48 hours. The daphnids were considered immobile when they were not able to swim for 15 seconds after gentle agitation of the test vessel. In addition to immobility, sub-lethal effects such as floating at the surface, were recorded.

The daphnids were randomly placed in the test solutions and the test vessels were positioned in a random manner. During the test the vessels were covered with glass plates. The test solutions were not aerated. The animals were not fed during the test.
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.04 mg/L
Remarks on result:
other: IUCLID4 note: "m" (measured/nominal)
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
0.16 mg/L
Remarks on result:
other: IUCLID4 note: "m" (measured/nominal)
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.04 mg/L
Remarks on result:
other: IUCLID4 note: "m" (measured/nominal)
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.077 mg/L
Remarks on result:
other: IUCLID4 note: "c" (calculated)
Reported statistics and error estimates:
The EC50 was calculated with the computer program TOXCALC version 5.0 using the trimmed Spearman-Kärber method. The concentrations causing zero and 100% immobility were derived directly from the test observations.
RESULTS: EXPOSED
- Nominal/measured concentrations: 0, 0.01, 0.02, 0.04,
0.08, 0.16 mg/L (nominal)
- Effect data (Immobilisation):
Immobile Daphnia
conc mg/L 24 hrs 48 hrs
0 0 1
0.01 0 2
0.02 1 2
0.04 0 1
0.08 0 11
0.16 9 20

The EC50 - 48 hrs was calculated to be 0.078 mg/L with 95% confidence limits of 0.066 and 0.092 mg/L. At 24 hrs no
effects were too low to derive an EC50. The NOEC 48 h amounted to 0.04 mg/L, the LOEC 48 h was 0.08 mg/L. The
EC100 was 0.16 mg/L and was observed after 48 hrs.
- Concentration / response curve: -
- Cumulative immobilisation: see Effect data
(Immobilisation)
- Effect concentration vs. test substance solubility: -
- Other effects: Sublethal effects were not observed in the
test (concentrations where no immobility was observed).
RESULTS CONTROL: see Effect data (Immobilisation)
Validity criteria fulfilled:
yes
Conclusions:
Study peformed according to standard protocol and under GLP.

Validity criteria can be considered as fulfilled, immobilisation of control animals was <10%, the control animals were not staying at the surface,
the concentration of dissolved oxygen in all test vessels was >3 mg/L and the concentration of test substance >= 80% of initial concentration
during test.
Although quantification of the applied concentrations was not possible due to the absence of a suitable analytical method. Therefore only the stock solution used to prepare the test concentrations were analyzed. These analyses showed that the stock solution was stable even after storage
for almost 7 months in the refrigerator. Considering the high water solubility of the test substance and its stability it is reasonable to assume
that the actual concentrations were identical to the nominal concentrations during the entire test.

A dose-response related increase in mortality was observed.

Under the circumstances of this test the LC50 (48 h) in Daphnia magna was calculated to be 0.0781 mg/L with 95% confidence limits of 0.066
and 0.092 mg/L.
Executive summary:

The acute toxicity of Triameen Y 12 D to Daphnia magna (water flea) was tested under static conditions for 48 hours in accordance with OECD and EEC guidelines for testing of chemicals. The test was performed in compliance with Good Laboratory Practice (GLP). The daphnia were exposed to the following nominal test concentrations: 0.01 – 0.02 – 0.04 – 0.08 and 0.16 mg/L.The EC50 (48 h) was calculated to be 0.078 mg/L with 95% confidence limits of 0.066 and 0.092 mg/L. The EC100 was 0.16 mg/L, which was observed after 48 hours. Sublethal effects were not observed in the test concentrations where no immobility was observed.

Quantification of the applied concentrations was not possible due to the absence of a suitable analytical method. The currently available analytical method is capable of analyzing down to only 10 mg/L. Therefore only the stock solution used to prepare the test concentrations was analyzed. These analyses showed that the stock solution was stable even after storage for almost 7 months in the refrigerator. All concentrations mentioned in this report are therefore based on nominal concentrations. Considering the high water solubility of the test substance and its stability it is reasonable to assume that the actual concentrations were identical to the nominal concentrations during the entire test.

Description of key information

Only a limit number of short-term toxicity to aquatic invertebrates studies are available for the substances under consideration. Therefore the test results of a number of alkyl-1,3-diaminopropanes and for dodecyltriamine Y were added to the dossier. Tri- and tetramines contain respectively about 22 – 11% diamines and for this reason the data for the diamines was added to the overview.  It is considered reasonable to add dodecyltriamine Y as a worst-case as this substance is considered a the most toxic triamine. The study with dodecyltriamine Y is selected as the Key study as this study is a Klimisch 1. The LC50 of this study is therefore used as the Key value for chemical safety assessment as a reasonable worst-case. In the final risk assessment this value will not be critical as two chronic end points are available.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.077 mg/L

Additional information

Most of the short-term toxicity daphnia tests were conducted in a period when no reliable specific method of analyses was available. The concentrations were therefore not analytically verified, the reliability of the results is limited because of the poor solubility of the test substances and partial sorption onto the walls of test vessels. Hence, most of the tests were considered to be valid with restrictions. Only one acute daphnia test study is available with a Klimisch 1 which is for N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (2372-82-9; dodecyltriamine Y). This substanceis used as a biocide and this substance is considered as the most ecotoxic substance for the alkyl triamines (3N).The observed LC50 for this substance of 0.0775 mg/L, suggests that the alkyl-1,3-diaminopropanes (2N) are more ecotoxic than the alkyl polyamines (>2N). Tri- and tetramines contain respectively about 22 – 11% diamines and for this reason the data for the diamines was added to the list of tests.  For oleyl tetramine (4N) an EC50 (48 h) has been observed of 0.032 mg/L. This value is slightly lower than the value observed for dodecyltriamine Y. For oleyl triamine a long term test result is available with specific chemical analysis which is used for the hazard assessment because the results can be used for the bulk approach where the sorption to glassware was minimal and solution stability much better.