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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-06-04 to 2009-09-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Twice prior to the start of the definitive exposure, samples from alternating replicates of each concentration and the dilution water control solutions were sampled and analysed for the concentration of test substance present in each vessel. Results of these pretest analyses were used to judge whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the early life-stage exposure.

During the in-life phase of the definitive study, samples were removed from alternate replicates (A/B/C/D) of each treatment level and control on days 0, 5, 12, 19, 21, 26, 28, 33, 40, 47, 54, 61, 64, 68, 70, 76, 80, 82 and 90 for analysis of test substance concentration. Additional samples of the test solutions were collected at each sampling interval and stored frozen for possible analysis in the event that routine analytical results were variable. All test samples were removed from the approximate midpoint of each aquarium using a pipette.

Three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure solution samples through the analytical process. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
Vehicle:
yes
Details on test solutions:
A 500 μg a.i./mL diluter stock solution was prepared prior to test initiation and as needed during the study by placing, for example, 0.0505 g of the test substance (0.0500 g as active ingredient) in a 100-mL volumetric flask and bringing it to volume with dimethylformamide (DMF, CAS No. 68-12-2). The resultant stock solution was observed to be clear and colourless. A 770 μL/mL solvent stock solution was prepared by diluting 770 mL of DMF to a final volume of 1000 mL with deionised water. The resulting stock solution was observed to be clear and colourless.

Prior to the pretest period, a Harvard Apparatus syringe pump in conjunction with a 100-mL Glenco® gas-tight syringe was calibrated to deliver 0.0660 mL/cycle of the 500 μg a.i./mL stock solution into the diluter system's chemical mixing chamber which also received 1.94 L of dilution water per cycle. The mixing chamber was positioned over a magnetic stir plate and was partially submerged within an ultrasonic water bath which aided in the solubilisation of the test substance into the dilution water. The concentration of test substance in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (17 μg a.i./L) and was proportionally diluted (50%) to produce the remaining nominal test concentrations (8.5, 4.3, 2.1 and 1.1 μg a.i./L).

An FMI pump was calibrated to deliver 0.234 mL/cycle of the 770 μL/mL solvent stock solution to 5.30 L of dilution water per cycle which was subsequently delivered to the solvent control vessels. The concentration of DMF in the solvent control vessels was equivalent to the concentration of solvent present in all treatment level solutions (34 μL/L). A set of control vessels was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, however, contained no test substance or solvent.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM

- Common name: Rainbow trout

- Source: The "green" rainbow trout eggs and sperm used during this study were obtained from TroutLodge, Inc., a commercial supplier in Sumner, Washington.

- Receipt and handling prior to test: On the day of test initiation, two bags containing the unfertilised gametes were received from TroutLodge, Inc., Sumner, Washington. These bags were received at a temperature of approximately 8 ºC and were warmed in a water bath to 11 ºC gradually over a one-hour period. The unfertilised eggs were approximately 30 hours old upon receipt and were fertilised within 2 hours of arrival.

- Fertilisation of eggs: Fertilisation was achieved by adding cold “fertilisation buffer” solution to the bag containing the eggs. The bag was rocked gently back and forth a few times and then placed in a water bath at test temperature (11 ºC) for approximately 20 minutes. This procedure was used to rinse away any ovarian fluid sealing the eggs, preventing fertilisation. Following the rinsing procedure, the eggs were removed from the water bath and most of the buffer solution was poured off. Sperm and an aliquot of buffer solution were added to the bag containing the eggs. The bag was then rocked gently back and forth and placed in a water bath for 15 minutes to complete the fertilisation process. The sperm was then poured off of the eggs and the eggs were then gently rinsed of excess sperm using cold dilution water. The eggs were then placed in a stainless steel bowl containing cold dilution water and allowed to water-harden for approximately 1.5 hours. During the water hardening period, the eggs were maintained at a temperature of 11 ºC.


POST-HATCH FEEDING

Larvae were fed a diet consisting of live brine shrimp nauplii (Artemia salina) three times daily beginning on day 9 post-hatch, when the rainbow trout began to exhibit swim-up behaviour. Larvae were not fed during the final 48 hours prior to test termination.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
90 d
Remarks on exposure duration:
60 days post hatch
Hardness:
Total hardness and alkalinity ranges as CaCO3: 48 to 64 mg/L and 18 to 26 mg/L, respectively
Test temperature:
10-14ºC
pH:
6.5-7.8
Dissolved oxygen:
6.6-11 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control), 1.1, 2.1, 4.3, 8.5 and 17 μg a.i./L

Arithmetic mean measured concentrations: 0 (Control), 0 (Solvent control), 0.92, 2.0, 4.0, 7.8 and 14 μg a.i./L

The results are reported and interpreted with reference to arithmetic mean measured concentrations.
Details on test conditions:
The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967) and a set of 28 exposure vessels. The exposure system was designed to provide five concentrations of the test substance, a dilution water control and a solvent control to four replicate aquaria.

Test vessels were impartially placed in a water bath containing circulating water designed to maintain the test solution at a temperature of 12 ± 2 ºC.
The concentration of test substance in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (17 μg a.i./L) and was proportionally diluted (50%) to produce the remaining nominal test concentrations (8.5, 4.3, 2.1 and 1.1 μg a.i./L).

Dimethylformamide (DMF) was used as a co-solvent. The concentration of DMF in the solvent control vessels was equivalent to the concentration of solvent present in all treatment level solutions (34 μL/L).

Dissolved oxygen concentration, pH and temperature were measured in all test vessels on test day 0 and in alternating replicates daily thereafter. Total hardness and total alkalinity as CaCO3, and specific conductance were measured on day 0 and weekly thereafter in alternating replicates of the low and high concentration and the dilution water control.

Testing was conducted during a 90-day (60-day post-hatch) exposure period, at a temperature range of 10 to 14 ºC, a photoperiod of 16 hours of light at 56 to 80 footcandles (610 to 860 lux) and 8 hours darkness.

On test day 19, mean embryo viability determined in the controls and all treatment levels tested ranged from 68 to 81% and was consistent with expectations and historical performance.

At termination of the early life-stage exposure, data obtained on embryo hatching success, percent normal larvae at hatch, larval survival and larval growth (total length and dry weight) were statistically analysed to establish treatment level effects. Results of this study are based on arithmetic mean measured concentrations.

The dilution water used during this study was unadulterated water from a 100-meter bedrock well which was pumped into an epoxy-coated concrete reservoir where it was aerated prior to use. During the study, weekly characterisation of the well water established total hardness and alkalinity ranges as CaCO3 of 38 to 60 mg/L and 17 to 24 mg/L, respectively, a pH range of 6.3 to 7.3 and a specific conductivity range of 320 to 380 μmhos/cm. Representative samples of the dilution water source were analysed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected in any of the water samples analysed at concentrations that are considered toxic, in agreement with ASTM (2005) standard practice. In addition, representative samples of the dilution water source were analysed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water ranged from 0.36 to 0.49 mg/L for the period of June to September 2009.
Reference substance (positive control):
no
Duration:
90 d
Dose descriptor:
NOEC
Effect conc.:
>= 14 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching, survival and growth
Duration:
90 d
Dose descriptor:
LOEC
Effect conc.:
> 14 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching, survival and growth
Reported statistics and error estimates:
At the termination of the early life-stage exposure, data obtained on embryo hatching success, percent normal larvae at hatch, larval survival and larval growth (total length and dry weight) were statistically analysed to establish treatment level effects. Analyses were performed using the mean organism response in each replicate aquarium rather than individual response values. All statistical analyses were conducted at the 95% level of certainty except in the case of the Bartlett's Test and Shapiro-Wilks' Test, in which the 99% level of certainty was applied.

TOXSTAT® Version 3.5 (West and Gulley, 1996) was used to perform the statistical computations. The lowest arithmetic average measured concentration that elicited a statistically significant effect on organism performance (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that elicited no statistically significant difference between the exposed organisms and the control (No-Observed-Effect Concentration, NOEC) were determined. Determination of these levels is based on the most sensitive of the performance criteria evaluated (e.g., embryo hatching success, percent of embryos that produce live normal larvae at hatch, organism survival at hatch, larval survival and growth at study termination).

Table 1. Results of analysis of test media

 Nominal concentration (μg/L) Arithmetic mean measured concentration (μg/L) (SD)  N  Mean percentage of nominal
 1.1  0.92 (0.16)  14  84
 2.1  2.0 (0.35)  14  95
 4.3  4.0 (0.67)  14  93
 8.5  7.8 (1.3)  14  91
 17  14 (2.3)  14  82

Table 2. Test results

 Mean measured concentration  (μg/L)  Mean percentage hatch  Mean percentage larval survival to end of test  Mean total length at end of test (mm) (SD) Mean dry weight at end of test (g) (SD)
 0 (Control)  82  92  52.5 (0.65)  0.2394 (0.0132)
 0 (Solvent control)  89  90  52.1 (0.42)  0.2382 (0.0171)
 0 (Pooled control)  86  91  52.1 (2.85)  0.2388 (0.0008)
 0.92  94  92  52.0 (0.96)  0.2399 (0.0174)
 2.0  90  92  51.2 (1.02)  0.2317 (0.0164)
 4.0  90  90  51.7 (1.05)  0.2398 (0.0157)
 7.8  83  90  51.6 (0.96)  0.2358 (0.0152)
 14  89  92  51.7 (0.97)  0.2460 (0.0176)
Validity criteria fulfilled:
yes
Conclusions:
A 90-day (60-day post-hatch) NOEC of ≥14 μg/L has been determined for the effects of the test substance on hatching and larval survival and growth of Oncorhynchus mykiss under flow-through exposure conditions.

Description of key information

90 day NOEC ≥14 μg/l, measured, Oncorhynchus mykiss (Springborn Smithers 2009f). This is above the water solubility of the registration substance D6 (5.1 µg/l); therefore, D6 is not expected to show toxicity at its limit of solubility.

Key value for chemical safety assessment

Additional information

Reliable test results are available for long-term toxicity to fish. A 90-day Read-Across NOEC of ≥14 µg/L has been determined for the effects of the read-across substance decamethylcyclopentasiloxane (D5, CAS 541-02-6) on hatching and larval survival and growth of Oncorhynchus mykiss. The results were obtained from an early-life stage: reproduction, (sub)lethal effects flow-through test, conducted in accordance with OECD Guideline 210 and in compliance with GLP (Springborn Smithers 2009f). In view of the exposure regime it is likely that the test organisms were exposed primarily to the tested substance rather than its degradation products.

The read-across justification is discussed in the Ecotoxicological Information endpoint summary in Section 6 of IUCLID, Section 7.0 of the CSR and in the siloxanes category supporting report (PFA 2017).

This is supported by a 49-day NOEC of >4.4 μg/l for the effects of the registration substance, D6, on mortality of adult fish (Pimephales promelas) (Dow Corning 2005). No other effects were assessed in this test, which was primarily designed to study bioconcentration.

The conclusion of no effects at the limit of solubility is further supported by a peer-reviewed study by Redman et al. (Redman 2012). A tissue based risk assessment was performed, with measured tissue concentrations of D4, D5 and D6 compounds in fish and benthic invertebrates compared with critical target lipid body burdens (CTLBBs) as estimated with the target lipid model (TLM). Acute and chronic toxicity data for these compounds were compared with data for nonpolar organic chemicals to validate application of the TLM in this effort. The analysis was extended to estimate the contribution from metabolites to the overall tissue residues using a food chain model calibrated to laboratory and field data. Concentrations of the compounds in biota from several trophic levels, including fish, are well below the estimated CTLBBs associated with acute and chronic effects. The data support the use of the TLM model (which assumes a non-polar narcosis mode of action) for D4, D5 and D6 and demonstrate their generally low bioavailability.

References

PFA, 2017, Siloxane Category Report for Environmental Endpoints, PFA.404.114.001.

Redman, A. D., Mihaich, E., Woodburn, K., Paquin, P., Powell, D., McGrath, J. A. & Di Toro, D. M. (2012). Tissue-Based Risk Assessment of Cyclic Volatile Methyl Siloxanes. Environmental Toxicology and Chemistry 31, 1911–1919.

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