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EC number: 220-562-2
CAS number: 2814-77-9
Pigment Red 4 is a red powder with a water solubility of approximately 3 µg/L. Three loading rates were tested for profound estimation of the NOELR and LOELR values. According to the guideline the highest loading rate did not exceed 10 mg/L.
With regard to the specific properties of the test item (small particles, poorly water soluble, pigment), the study was carried out with dispersions of the test item (i.e. in excess of water solubility). The preparation of the dispersions included ultra-sonication and vigorous mixing to disperse the test item in water, followed by low energy mixing with circulating pumps to prevent sedimentation. The study was conducted as a semi-static test and daily renewal of the test media. Daily renewal was chosen, due to the outcome of the preliminary experiments on dispersion stability carried out referring to OECD 318.
The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio / zebrafish were exposed to each loading rate and the control (4 replicates with 20 eggs each).
The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.
On study day four, 98 % of the control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).
Different toxicological endpoints were determined: hatching success, time to hatch, fry growth (expressed as length and fresh weight), morphological and behavioral effects, post-hatch survival and overall survival (mortality).
Specific analysis of each loading rate of Pigment Red 4 and the control was carried out via LC-MS/MS. The dispersed fraction of the test item was determined after addition of DMSO directly after sampling. The dissolved fraction was determined after centrifugation of the water samples, to remove undissolved test item. The concentrations were analytically verified twice during 7 days of exposure from freshly prepared test media on study day 0 (start of the exposure), 7, 12, 21 and 28 and from corresponding 24 hours aged test media on study days 1, 8, 13, 22 and 29.
As the test item disperses, the effect values are considered to be based on the nominal loading rates of 1.00 – 3.16 – 10.0 mg/L, corresponding to the geometric mean measured concentrations of the dispersed fraction of the test item 0.340 – 1.16 – 2.75 mg/L and the geometric mean measured concentrations of the dissolved fraction of the test item 0.0200 – 0.0401 – 0.0413 mg/L, respectively.
All effect levels were given based on the geometric mean measured concentrations of the total fraction of the test item and on the geometric mean measured concentrations of the dissolved fraction of the test item. In the samples, the sedimentated fraction was not included. However, this fraction was also present in the test vessels.
Findings and Observations
The results of the parameters hatching success, time to hatch, fry growth (expressed as weight and length), post-hatch survival and overall survival were checked for statistically significant differences.
No statistically significant differences were detected between the dilution water control and each loading rate of the test item. In conclusion, under the conditions of this test the test item did not affect early-life stages of the zebrafish at a nominal loading rate of 10 mg/L.
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