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Isononyl benzoate was tested for potential toxicity towards aquatic organisms in fish (acute and chronic toxicity), daphnia (acute and chronic toxicity) and algae (acute/chronic toxicity). Furthermore its effects on the microbial community of activated sewage sludge was investigated to assess potential effects of emissions to sewage treament plants. The test item is poorly soluble in water and in the acute toxicity studies no adverse effects on the exposed organisms were recorded up to the solubility limit.

Isononyl benzoate was tested in acute toxicity with Cyprinus carpio under semi-static conditions according to the guidelines EEC specification 92/69/EEC (C.1) and OECD 203. The test was conductet as limit test. In order to attain the solubility limit of the test substance, which is poorly soluble in water, 1g test substance/ l was stirred for 18 h and undissolved test substance was then removed by filtration. A loss of test substance over the test period was recorded by measurement of dissolved organic carbon, the concentration values given are therefore mean measured values. No effects on the exposured carps were recorded, thus the LC50 > 1.23 mg/l which constitutes the solubility limit of isononyl benzoate under the conditions of the test.

The effects of the test item Isononyl benzoate to the early-life stage of fish (Zebrafish Danio rerio) were determined according to OECD guideline 210. A test was conducted under flow-through conditions with the nominal concentrations 62.5 - 125 - 250 - 500 - 1000 µg/L, corresponding to mean measured concentrations of 19.3 – 42.8 – 90.5 – 297 – 538 µg/L. The test was started by placing fertilised eggs in the test vessels and lasted 33 days (28 days post-hatch). 60 eggs of Danio rerio were exposed to the test concentrations and the control (4 replicates with 15 eggs each). Water quality parameters pH-value, oxygen concentration, temperature, hardness were determined to be within the acceptable limits. On day five 95 % of the control larvae had hatched. Therefore, study day 5 had been defined as post hatch day 0 (= PHD 0). Different toxic endpoints have been determined: egg hatch, time to hatch, fry growth (expressed as length and weight), morphological and behavioural effects, post hatch survival and fry survival. Chemical analysis (HPLC) of the test solutions was carried out in at least weekly intervals. Due to the test item properties adsorption and biodegradation were expected to occur. Therefore a flow rate equivalent to twenty test chamber volumes per 24 hours (guideline recommendation at least 5) had been set up in order to minimize absorbance and biodegradation effects. However, the mean recovery rates were in the range of 31 - 59 % of the nominal concentrations. For the lowest concentration most of the measurements were below the LOQ (0.03 mg Isononylbenzoat/L). With regard to the test item properties the analytical results were considered not to invalidate the test. All effect levels will be stated on base of both nominal and mean measured concentrations.

Findings and Observations Based on the observation and statistical analysis of egg hatch, time to hatch, growth (expressed as weight and length), morphological and behavioural effects, post hatch survival and fry survival, the test revealed the following results:

Table1:  Hatch, Growth, Morphological Effects, Fry survival: NOEC, LOEC

                  Based on nominalIsononylbenzoatconcentrations and mean measured concentrations in brackets, respectively.

Hatch:

Hatch on study days 3 to 5 resulted in a NOEC of³1000(538) µg/L and a LOEC of > 1000 (538) µg/L.

Growth1)

Length on PHD 28 resulted in a NOEC of ³ 250 (³ 90.5) µg/L and a                       LOEC of > 250 (> 90.5) µg/L1).  

Dry weight on PHD 28 resulted in a NOEC of  ³ 250 (³ 90.5) µg/L and a            LOEC of > 250 (> 90.5) µg/L.1)

Morphological and behavioural effects

Morphological and behavioural effects resulted in a                                         NOEC of³ 250(³ 90.5) µg/L and a LOEC > 250 (> 90.5) µg/L.

Post hatch survival

Post hatch survival on PHD 28 resulted in a NOEC of 250 (90.5) µg/L and a LOEC 500 (90.5) µg/L.

Overall survival

Overall survival on PHD 28 resulted in a NOEC of 125 (42.8) µg/L and a LOEC of 250 (90.5) µg/L.

Overall NOEC and LOEC

Based on the parameters hatch, growth (expressed as length and dry weight), post hatch and overall survival the overall NOEC (post hatch day      0 – 28) was 125 µg/L (42.8µg/L) and the overall LOEC was 250 µg/L (90.5 µg/L).

1) The concentrations 500 µg/L and 1000 µg/L were not considered to statistical evaluation due high mortality and reduced fish density in these replicates (number of surviving fish 80 % of the control).

Isononyl benzoate was tested in acute toxicity test with daphnids (Daphnia magna Straus) under static conditions according to the guideline EU Method C.2 and OECD Test Guideline 202 (Acute Toxicity for Daphnia). The test was conducted as limit test. In order to attain the solubility limit of the test substance, which is poorly soluble in water, 1 g test substance /L was stirred for 18 h and undissolved test substance was then removed by filtration. The measured test concentration after 48 h deviated by more than 20% from the freshly prepared test solution. Therefore, the biological value was calculatet with the geometrical mean of the analytical concentration after 0 and 48 hours. No effects on the exposed daphnids were recorded, thus the EC50 is > 2.2 mg/L which constitutes the solubility limit of isononyl benzoate under the conditions of the test.

The Daphnia magna STRAUS Reproduction Test (21d) of isononyl benzoate was conduced as a limit test with the saturated solution* at the range of water solubility according to OECD 211 (1998) and Directive 2001/59/EC Method C.20 (2001). The saturated solution is the maximum dissolved concentration of the test item that can be achieved under the conditions of the slow stirring method in the test medium, acc. to OECD Series, No.23 (2000)6.

The Test system was Dapnia magna STRAUS (Clone 5). 10 test organisms, individually held were used for the limit concentration and the control. At test start the daphnids were 2 to 24 hours old. The test method was semi-static. Test solutions wer renewed daily. Aim of the test was to assess effects on the reproduction rate and other test item-related effects on reproduction parameters such as occurrence of aborted eggs, stillborn juveniles, males and ephippia, time of release of first brood, number of broods, adult mortality and the intrinsic rate of natural increase. The test concentration was verified analytically by HPLC. The NOEC (21d) > = 0.078 mg/L, which constitutes the limit of solubility of the test item under the test condition of this study. No mortality and no adverse effects on reproduction were observed. On the contrary, the reproduction rate of the exposed daphnids showed a significant increase compared to the control values.

Isononyl benzoate was tested in growth inhibiton test with single cell green algae Desmodesmus subspicatus under static conditions according to the guidelines EU Method C.3 and OECD 201. 1 g / l test substance was stirred for 18 h and undissolved test substance was then removed by filtration. All samples analysed (incl.the stock solution) were below the limit of quantification of the analytical method, i.e. < 1 mg TOC/l. Therefore, no analyses were performed on the test concentrations after 72 h. No negative effects on growth rate were recorded, thus the NOEC growth rate is > = the limit of solubility of test item under test conditions and the EC50 growth rate is > the limit of solubility (limit of detection of TOC analysis = 1 mg/L). In the concentration range tested, the test item had a positive effect on the growth of the algae.

Isononyl benzoate was tested for its potential to inhibit microbial activities and therefore potentially interfere with sewage treatment plant microbial communities in a GLP study according to OECD 209. As test organism activated sludge from the sewage treatment plant Marl-West, predominantly treating domestic sewage was used. The contact time was 3 hours. No inhibition of the activated sludge respiration was observed up to the highest concentration tested. Thus, the EC20, EC50 and EC80 > 1000 mg/L.