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Long-term toxicity to fish

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Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-07-22 to 2010-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
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 L3 present in each vessel. Results of these pre-test 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, 4, 13, 20, 27, 33, 35, 40, 47, 54, 61, 63, 68, 74, 76, 79, 82 and 90 for analysis of L3 concentration. The results for samples analysed on days 33, 74 and 76 were not used due to issues associated with processing of samples. All test samples were removed from the approximate midpoint of each aquarium using a syphon.

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:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Concentrations: Based on the information provided by and in agreement with the Study Sponsor, nominal L3 concentrations of 2.1, 4.3, 8.5, 17 and 34 μg a.i./L were selected for the definitive early life-stage exposure. The reported water solubility for L3 is 34 μg/L.

- Stock solutions: A 2.0 mg a.i./mL diluter stock solution was prepared prior to exposure initiation and as needed during the study by placing 0.2001, g of L3 (0.2000 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 560 μL/mL solvent stock solution was prepared by diluting 560 mL of DMF to a final volume of 1000 mL with deionized water. The resulting stock solution was observed to be clear and colourless.

- Diluter system: Prior to the pre-test period, a Harvard Apparatus syringe pump in conjunction with a 100-mL Glenco® gas-tight syringe was calibrated to deliver 0.03298 mL/cycle of the 2.0 mg 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 solubilization of the test substance into the dilution water. The concentration of L3 in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (34 μg a.i./L) and was proportionally diluted (50%) to produce the remaining nominal test concentrations (17, 8.5, 4.3 and 2.1 μg a.i./L).

The concentration of DMF in the solution in the mixing chamber and the high test concentration constituted the highest DMF concentration (17 μL/L). An FMI pump was calibrated to deliver 0.166 mL/cycle of the 560 μL/mL solvent stock solution to 5.51 L of dilution water per cycle which was subsequently delivered to the solvent control vessels. The DMF concentration in the solvent control and the treatment levels was 17 μL/L, which was equal to that of the high test concentration.

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, but contained no L3 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., Sumner, WA, USA

- Test fish (age/length/weight, loading, pre-treatment): Newly fertilized eggs (≤1 hr postfertilization)

- Treatment prior to exposure: The bags containing the eggs were received at a temperature of approximately 8 ºC and were warmed in a water bath to 10 ºC gradually over a one-hour period. Fertilization was achieved by adding cold fertilization buffer solution, consisting of 3.75 g/L glycine, 2.42 g/L TRIS and 5 g/L sodium chloride, 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 (approximately 10 ºC) for approximately 20 minutes. This procedure was used to rinse away any ovarian fluid sealing the eggs, preventing fertilization. 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 20 minutes to complete the fertilization 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 30 minutes. During the water hardening period, the eggs were maintained at a temperature of 10 ºC.

The eggs were then impartially distributed to the egg incubation cups in the following manner: twenty-eight labelled egg incubation cups were set in a shallow water bath maintained at 10 ºC. The collected eggs were then counted into each egg cup, sequentially, five at a time, until each cup contained five eggs. This procedure was repeated until each egg cup contained 30 eggs. To initiate the exposure, the incubation cups, each containing 30 eggs, were then placed on the rocker arm apparatus in the respective exposure aquarium (one cup per replicate vessel). The eggs were approximately one hour old (post-fertilization) at exposure initiation.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
90 d
Remarks on exposure duration:
(approximately 6 weeks after controls were free-feeding)
Hardness:
56 – 76 mg/L as CaCO3
Test temperature:
8.8-10°C for embryo incubation period

11-13°C for larval growth period
pH:
6.6 –7.6
Dissolved oxygen:
3.4 – 13 mg/L (31 - 107% of saturation)

Dissolved oxygen levels <60% saturation were not observed for >8 hours.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent Control), 2.1, 4.3, 8.5, 17 and 34 μg/L

Time-weighted mean measured concentrations:
The results are interpreted with reference to time weighted mean measured concentrations
Details on test conditions:
- Details of apparatus: The study was conducted using an exposure system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath and a set of 28 exposure aquaria. Flow-splitting cells were employed to equally distribute the solutions to the four replicate vessels at a rate of approximately 250 mL of test solution per vessel per cycle. Flow splitting accuracy of the diluter cells was within 5% of the nominal value. The test system was designed to provide five concentrations of the test substance, a solvent control and a dilution water control to four replicate aquaria.

The diluter system and exposure aquaria were fabricated of glass and silicone sealant. Each test aquarium measured 30 x 15 x 20 cm with a 14.5-cm high side drain that maintained a constant exposure solution volume of 6.5 L. Embryo incubation cups were round glass jars (5 cm) diameter, 8 cm high with 1000-micron mesh opening Nitex® screen bottoms. A rocker arm apparatus, driven by a 2 rpm electric motor was used to gently oscillate the incubation cups in the test solutions. Test aquaria were labelled to identify the treatment levels, control and designated replicate (A, B, C or D).

The exposure system was properly operating for at least 48 hours prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure aquaria and to monitor the behaviour of the test substance over time.

Eggs were impartially distributed to the egg incubation cups in the following manner: twenty-eight labelled egg incubation cups were set in a shallow water bath maintained at 10 ºC. The collected eggs were then counted into each egg cup, sequentially, five at a time, until each cup contained five eggs. This procedure was repeated until each egg cup contained 30 eggs. To initiate the exposure, the incubation cups, each containing 30 eggs, were then placed on the rocker arm apparatus in the respective exposure aquarium (one cup per replicate vessel). The eggs were approximately one hour old (post-fertilization) at exposure initiation. After hatching the number of fish in each test vessel was reduce to 15.

- Feeding: Larvae were fed a diet consisting of live brine shrimp nauplii (Artemia salina) three times daily beginning on day 14 post-hatch, when the rainbow trout began to exhibit swim-up behaviour. At each feeding, larvae were fed ad libitum such that all larvae were afforded equal access to food. Larvae were not fed during the final 48 hours prior to test termination.

- Dilution water source: unadulterated well water. Hardness 50 – 86 mg/L as CaCO3, alkalinity 17 – 22 mg/L as CaCO3 and conductivity 360 – 630 μmhos/cm

- Test conditions: Test aquaria were impartially positioned in a water bath containing circulating water cooled by two Frigid Unit® 3/4-horsepower chillers which were designed to maintain the test solution temperature at 10 ± 2 ºC during the embryo incubation period and 12 ± 2 ºC during the larval grow out period. During the embryo incubation phase and larval development phase prior to the swim-up stage, the test area was maintained in continuous darkness. Once all larvae had reached the swim-up stage, a 16-hour light photoperiod at a light intensity range of 73 to 120 foot candles (790 to 1300 lux) and 8 hours darkness with a 30-minute transition period was provided at the exposure solutions' surface. Illumination was provided by Sylvania Octron® fluorescent lights centrally located above the test aquaria. Light intensity was measured with a VWR Traceable light meter. At the swim-up stage, screens were placed on the tops of the test aquaria to prevent the fish from escaping.

- Flow-through rate: The diluter delivered the control and test solutions to the exposure aquaria (97 L/aquarium/day) at a rate sufficient to provide approximately 15 aquarium volumes per 24-hour period, with a 90% replacement time of approximately three hours.

Observations: Due to the extreme sensitivity to physical trauma during the early developmental stage, dead eggs were not removed from the egg cups prior to reaching the "eyed" developmental stage of the exposure period. A definitive determination of viability was made on day 18, when all the eggs were passed the physically sensitive stage and exhibited well-pronounced embryonic development. Unfertilized eggs and dead eggs, characterized by an opaque, white colour, were removed after this time and preserved in Stockard's solution (an 85:6:5:4 mixture of water, glycerine, formalin, and glacial acetic acid), which also served to clear the eggs, enabling a determination of embryonic development. Any egg exhibiting embryonic development (up to and including day 18), whether dead or alive, was considered fertile for purposes of determining percent viability. All non-viable eggs were counted and discarded. Percent viability was based on the number of fertile embryos produced from the initial number of eggs exposed. The study continued by impartially selecting 15 live, viable embryos from each respective incubation cup and reassigning them into separate incubation cups which were suspended in the respective exposure aquaria. The larvae which hatched from these 15 isolated embryos were used to initiate the post-hatch phase of the exposure. The remaining viable embryos were placed in a second incubation cup and also suspended in the respective exposure aquaria. The remaining embryos were used to determine percent hatching success. This procedure assured unbiased thinning of larvae at the completion of hatch.

The day of hatch was defined as day 35, when no more than 10% unhatched viable embryos remained in any embryo incubation cup, thus initiating the 55-day post-hatch larval exposure, and providing a theoretical total of 15 larvae per replicate aquaria. The larvae from the second incubation cup were counted, euthanized with MS-222 (tricaine methane-sulfonate) and discarded. Calculations of hatching success of organisms were based on the number of live normal larvae per incubation cup after hatching was complete (day 35) compared to the number of embryos per cup on day 18 (thinning of eggs).

At 55 days post-hatch, the exposure was terminated. The surviving larvae in each replicate aquarium were euthanized with MS-222 (tricaine methanesulfonate), counted to determine larval survival, measured individually to determine the total length, and dried in an oven at 100 ºC for approximately 19 hours. Following the drying period, the larvae were individually weighed to determine dry weight. At termination, individual wet weights were also determined for the control larvae to determine biomass loading. The larvae were measured and weighed to the nearest 0.01 mm and 0.1 mg, respectively. The mean and standard deviation were calculated using individual measurements (total length and dry weight) for each replicate aquarium.

- Water quality: 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.
Reference substance (positive control):
no
Duration:
90 d
Dose descriptor:
NOEC
Effect conc.:
>= 27 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: embryo survival, larval hatch and growth (length and dry weight)
Duration:
90 d
Dose descriptor:
LOEC
Effect conc.:
> 27 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: embryo survival, larval hatch and growth (length and dry weight)
Details on results:
- Biological observations: There were no treatment-related observations of sublethal effects.

- Mortality of controls: 6%

- Any observations, such as precipitation that might cause a difference between measured and nominal values: None
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.

Table 1. Test results

Nominal concentration (μg/L)

Time-weighted mean measured

concentration

(μg/L)

 

Time-weighted mean measured

concentration

as % of nominal

Embryo

Survival

(%)

 

Normal

Larvae at

Hatch (%)

Larval

Survival

(%)

Total Body

Length

(mm)

 

 

Dry Body

Weight

(g)

0 (Control)

-

 

98

100

93

53.6

0.273

0 (Solvent control)

-

 

99

100

95

53.9

0.272

0 (Pooled control)

-

 

98

100

94

53.8

0.273

2.1

1.7

80

93

98

87

54.5

0.290

4.3

3.5

82

98

100

100

53.3

0.260

8.5

7.3

86

95

100

92

54.0

0.279

17

15

86

99

100

92

53.9

0.273

34

27

79

98

100

95

54.2

0.274

 There were no statistically significant treatment-related effects

Validity criteria fulfilled:
yes
Conclusions:
A NOEC of ≥27 μg/L has been determined in an early life stage test in which effects on embryo survival, larval hatch and growth (length and dry weight) of Oncorhynchus mykiss were evaluated over a 90-day period (including a period of approximately 6 weeks after controls were free-feeding). The results were obtained under flow-through test conditions and are expressed as time-weighted mean measured concentrations.
Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See endpoint summary for justification of read-across
Reason / purpose:
read-across source
Duration:
90 d
Dose descriptor:
NOEC
Effect conc.:
>= 27 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: embryo survival, larval hatch and growth (length and dry weight)
Duration:
90 d
Dose descriptor:
LOEC
Effect conc.:
> 27 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: embryo survival, larval hatch and growth (length and dry weight)

Description of key information

Long-term toxicity to fish: 90-d NOEC ≥0.027 mg/l (highest concentration tested) (OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)) read-across from read-across from an analogous/structurally related substance, octamethyltrisiloxane (CAS 107-51-7).

Key value for chemical safety assessment

Additional information

There are no reliable long-term fish toxicity data available for 1,1,1,3,5,5,5 -heptamethyltrisiloxane (CAS 1873 -88 -7), therefore good quality data for an appropriate structural analogue, octamethyltrisiloxane (CAS 107-51-7), have been read across.   A 90-d NOEC of ≥0.027 mg/l (time-weighted mean measured concentration) (highest concentration tested) have been determined for the effects of octamethyltrisiloxane (CAS 107-51-7) on embryo survival, larval hatch and growth (length and dry weight) of Oncorhynchus mykiss (including a period of approximately 6 weeks after controls were free-feeding). In view of the exposure regime it is likely that the test organisms were exposed predominantly to the tested substance. Refer to the discussion in the Section 6 endpoint summary (Section 7.0 of CSR) for further discussion of the approach to chemical safety assessment for this registration substance, and justification for read-across used.