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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:
14th of March 2020 to 15th of September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
July, 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Nominal concentrations: 0, 0.031, 0.063, 0.13, 0.25, 0.50 mg/L
Geometric mean measured concentration: 0, 0.0029, 0.0068, 0.011, 0.017, 0.036 mg/L
- Sampling method: Semi-static
- Sample storage conditions before analysis: Sample storage stability was not assessed.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
At the start of the test, groups of 20 eggs were added to glass scintillation vials half filled with treated mains water and each vial was randomly assigned to a test vessel.
The eggs were transferred using a discrete pipette into the test vessel. The eggs were released under the surface of the test media, as close to the bottom of the vessel as possible.
Observations were performed daily throughout the duration of the test and details of the following parameters were recorded:

• Hatching – number of larvae hatched until hatching was complete
• Egg mortalities – an egg was considered dead if it was observed to be cloudy or necrotic
• Fish mortalities – a larvae were considered dead if it did not respond to stimulus, via touch
• Sub-lethal effects including behavioural abnormalities (typical observations on appearance include size, paleness and any developmental effects such as spinal deformity or differences in body shape)
During the pre-hatch period, where possible, non-viable or necrotic eggs were removed, avoiding disturbance of adjacent viable eggs. The post-hatch phase started once all of the viable eggs were considered to have hatched in the control groups.

On Day 28 post-hatch, the total numbers of surviving larvae were counted and individual total fish lengths and wet weights of all remaining fish were determined. Prior to initial weighing, the fish were blotted dry to make sure that all surface water was removed.

The percentage post-hatch survival was determined by expressing the number of surviving larvae on Day 28 post-hatch as a percentage of the hatched larvae at Day 0 (post-hatch).
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead Minnow
- Strain: Pimephales promelas
- Source: From an in-house laboratory breeding system (Smithers ERS Limited)
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Weight at study initiation (mean and range, SD):
- Method of breeding:
- Feeding during test : The developing larvae were fed freshly cultured brine shrimp (Artemia salinis) nauplii. Surplus feed was siphoned from the test vessel on at least daily intervals during the test.
- Food type: Freshly cultured brine shrimp (Artemia salinis) nauplii.
- Amount: Twice daily, ad libitum.
- Frequency: Once hatching had started, the larvae were fed approximately 24-hour old shrimp. From Day 5 post-hatch and onwards, the larvae were fed approximately 48-hour old shrimp.

ACCLIMATION
- Acclimation period: At least one hour
- Acclimation conditions: According to laboratory conditions
- Type and amount of food: Not reported
- Feeding frequency during acclimation: Not reported
- Health during acclimation (any mortality observed): Not reported
Test type:
semi-static
Water media type:
freshwater
Remarks:
Treated mains water (treated via activated carbon particulate filter and ultraviolet irradiation).
Limit test:
no
Total exposure duration:
28 d
Remarks on exposure duration:
Duration was 13 days post-hatch.
Test temperature:
Water temperature: 23.5 to 25.4°C measured in the test vessels and 23.8 to 26.0°C for the continuously measured temperature.
pH:
7.21 to 7.84
Dissolved oxygen:
60.0 to 115.0% ASV (with one exception in a single vessel during the test which was 53.7%).
Nominal and measured concentrations:
Nominal concentrations: 0, 0.031, 0.063, 0.13, 0.25, 0.50 mg/L
Geometric mean measured concentration: 0, 0.0029, 0.0068, 0.011, 0.017, 0.036 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Size of vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Treated mains water (treated via activated carbon particulate filter and ultraviolet irradiation).
- Total organic carbon: No reported
- Particulate matter: Not reported
- Metals: various values between <0.009 to <0.23 mg/L
- Pesticides: <0.005 μg/L and <0.007 μg/L for tecnazene
- Chloride: 13.6 mg/L
- Alkalinity (as CaCO3): 10.8 mg/L
- Ca/mg ratio: 14.2/2.2 mg/L
- Conductivity (electrical 20°C): 145 μS/cm
- Culture medium different from test medium: N/a
- Intervals of water quality measurement: Water temperatures, pH and dissolved oxygen concentrations were measured at the start and end of the test. Fresh and corresponding old media water qualities were also conducted at least weekly for the duration of the test. Fresh media water qualities were conducted on the bulk media, whereas old media water qualities were conducted on individual replicates.

OTHER TEST CONDITIONS
- Adjustment of pH: Not necessary as the range was 7.29 to 7.49
- Photoperiod: 16-hour light: 8-hour dark period with an approximate 30-minute dawn:dusk transition period.
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED: N/a

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/a
- Justification for using less concentrations than requested by guideline: N/a

RANGE-FINDING STUDY
A solubility / stability trial was conducted in compliance with a separate GLP study to investigate the functional solubility of the test substance in dilution water and to determine the most appropriate dosing method. Based on the results, it was initially considered that the maximum functional solubility of the test substance in dilution water was 1.0 mg/L. Due to the limited functional solubility and instability of the test substance, it was believed that the semi-static exposure design would provide exposure to higher concentrations and a more consistent exposure pattern over the study period compared to flow-through design. Additionally, it was considered that the highest concentration of 1.0 mg/L used in the range-finding test was above the level of solubility of the test substance in dilution water. It was considered more realistic to have a concentration of 0.5 mg/L as this was estimated to be the maximum functional solubility of the test substance in treated mains water. Therefore, the final nominal concentrations were determined to be 0.031, 0.063, 0.13, 0.25 and 0.50 mg/L in the main test. Four replicates were prepared for the control, solvent control and each test concentration.

A range-finding test was conducted with a control, solvent control and nominal test substance concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L under semi-static conditions with daily renewal of the test media. Concentrations in excess of 1.0 mg/L were not tested at the request of the Sponsor as this was initially considered to be the limit of functional solubility of the test substance based on the solubility / stability trial. Single test vessels were prepared, with 10 eggs in each vessel, for the control, solvent control and each test concentration. The test duration was 13 days post-hatch.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: hatching success, normal larvae at hatch, posthatch survival and growth
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: hatching success, normal larvae at hatch, posthatch survival and growth
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.036 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching success, normal larvae at hatch, posthatch survival and growth
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 0.036 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching success, normal larvae at hatch, posthatch survival and growth
Details on results:
Analysis of the 4-hour old media on Day 0 (test start) showed a measured concentration of 68% of nominal for the 0.50 mg/L test concentration. This decreased to 33% of nominal after the first 24 hours of exposure (Day 1). On Day-0 post-hatch, the measured concentration in the 4-hour old media sample was 36% of nominal which decreased to less than the LOQ after 24-hours (Day-1 post-hatch). For the remainder of the test the 4-hour old sample results ranged from 8% to 24% of nominal with the 24-hour old samples generally less than the LOQ. These results showed the test substance was unstable in the test media but may also interact with biological material as the decrease in measured concentration over the 4- and 24hour periods increased as the organisms hatched and grew.

A similar trend in analytical measurements was observed for the remaining test concentrations.
Due to the decrease in measured concentration over each 24-hour exposure period, it was considered appropriate to base the results on geometric mean measured concentrations. These were calculated to be 0.0029, 0.0068, 0.011, 0.017 and 0.036 mg/L.

The mean hatching success of embryos in the control and solvent control groups group was 100% and 99%, respectively, which was in excess of the validity criterion of ≥70%. The mean post-hatch survival in the control and solvent control groups was 81% and 82%, respectively, which was in excess of the validity criterion of ≥75%. The test is therefore considered to be valid.

No statistically significant differences were observed in terms of hatching success, post-hatch survival, final fish lengths and final fish wet weights at any of the test concentrations employed in the test up to the test substance’s functional water solubility limit compared to the combined controls. In addition, no dose related effects were observed in terms of the number of normal larvae observed at hatch.

The limit of quantification (LOQ) was 0.0003 mg/L. Analysis of the freshly prepared media at a nominal concentration of 0.50 mg/L showed measured concentrations to range from 67% to 107% of nominal throughout the duration of the test. These results indicated that the test organisms were initially exposed to concentrations close to nominal at each media renewal.

Analysis of the 4-hour old media on Day 0 (test start) showed a measured concentration of 68% of nominal for the 0.50 mg/L test concentration. This decreased to 33% of nominal after the first 24 hours of exposure (Day 1). On Day 0 post-hatch, the measured concentration in the 4-hour old media sample was 36% of nominal which decreased to less than the LOQ after 24-hours (Day 1 post-hatch). For the remainder of the test the 4-hour old sample results ranged from 8% to 24% of nominal with the 24-hour old samples generally less than the LOQ. These results showed the test substance was unstable in the test media but may also interact with biological material as the decrease in measured concentration over the 4- and 24hour periods increased as the organisms hatched and grew.

A similar trend in analytical measurements was observed for the remaining test concentrations. Due to the decrease in measured concentration over each 24-hour exposure period, it was considered appropriate to base the results on geometric mean measured concentrations. These were calculated to be 0.0029, 0.0068, 0.011, 0.017 and 0.036 mg/L.
Reported statistics and error estimates:
Statistical analysis of the data was performed using the CETIS program v 1.8.6.8.
The hatching success results from the control and solvent control groups were compared using the Unequal Variance t Two-Sample test. The post-hatch survival, lengths and wet weight results were compared using the Equal Variance t TwoSample test. Based on this analysis, the results for the test concentrations were compared to the combined controls.

To determine the NOEC and LOEC, the number of hatched larvae were compared to the combined controls using the Dunn/Bonferroni-Holm Test. The number of larvae surviving at 28 days post-hatch and Day 28 lengths and wet weights for the test concentrations were compared to the combined controls using the Dunnett Multiple Comparison Test.

The NOEC and LOEC values for percentage normal larvae at hatch were not statistically derived given that only four abnormal larvae were observed at hatch and no concentration dependent response was observed.

No statistically significant differences were observed between the control and solvent control groups in terms of hatching success, post-hatch survival or growth (total lengths and wet weights). The results from the treatment groups were therefore compared to the combined control groups.

First egg hatch occurred between Day 2 and 4 and completion of hatching occurred between Days 4 and 6 (post-egg addition) in all treatment and the control vessels. NOEC and LOEC values for time to first egg hatch and the egg-hatching period were therefore not statistically derived. This indicated no difference in the time to first hatch or hatching period across all treatments when compared to the control and solvent control groups.

The mean hatching success of embryos in the control and solvent control vessels were 100% and 99%, respectively, which was in excess of the validity criterion of ≥70%. It was therefore considered that the validation criterion had been achieved. 

Mean hatching success in the treatments ranged between 80% - 90%. There were no statistically significant effects on hatching success at any of the test concentrations employed in the test compared to the combined controls.

Table 1 - Hatching success

Nominal concentration (mg/L)

Geometric Mean

Measured Concentration (mg/L)

Hatching success (%)

Control

Control

100

Solvent control

Solvent control

99

0.031

0.0029

85

0.063

0.0068

80

0.13

0.011

84

0.25

0.017

83

0.50

0.036

90

On Day 0 post-hatch, two larvae in the solvent control and a single larvae in the 0.031 and 0.50 mg/L test concentrations were observed to have a bent spine. No further abnormalities were observed in the newly hatched larvae. NOEC and LOEC values for percentage normal larvae at hatch were therefore not statistically derived. This indicated no difference in the percentage normal larvae at hatch across all treatments when compared to the control and solvent control groups.

Table 2 - Normal Larvae at Hatch

Nominal concentration (mg/L)

Geometric Mean

Measured Concentration (mg/L)

Normal larvae at hatch (%)

Control

Control

100

Solvent control

Solvent control

97

0.031

0.0029

99

0.063

0.0068

100

0.13

0.011

100

0.25

0.017

100

0.50

0.036

99

The mean post-hatch survival in the control and solvent control groups was 81% and 82%, respectively, which satisfied the validity criterion of ≥75%. It was therefore considered that the validation criterion had been achieved.

Mean post-hatch survival in the treatments ranged between 76% - 89%. There were no statistically significant effects on post-hatch survival at any of the test concentrations used compared to the combined control.

Table 3 - The Survival of Hatched Larvae at Day 28 post-hatch

Nominal concentration (mg/L)

Geometric mean measured concentration (mg/L)

Post-hatch survival Day 28

(%)

Control

Control

81

Solvent control

Solvent control

82

0.031

0.0029

76

0.063

0.0068

83

0.13

0.011

79

0.25

0.017

89

0.50

0.036

83

 

There were no statistically significant effects on fish length or wet weight at any of the test concentrations employed in the test compared to the combined controls.

Table 4 - Total Length (mm) and Wet Weight (mg) Measurements

Nominal concentration

(mg/L)

Geometric mean measured

concentration

(mg/L)

Mean total length (mm)

Mean wet weight (mg)

Control

Control

21.6

79.0

Solvent control

Solvent control

22.1

98.8

0.031

0.0029

22.9

109.6

0.063

0.0068

22.3

102.8

0.13

0.011

23.1

109.2

0.25

0.017

22.0

91.4

0.50

0.036

22.0

93.7

A small number of fish were observed to be either small, pale, to have a bent spine, to be swimming abnormally or to be small with a bent spine (see Appendix 5). However, these observations were not concentration specific and were therefore not considered to be due to any toxic effect but rather natural variation within the population. 

Validity criteria fulfilled:
yes
Conclusions:
In a long-term toxicity to fish study, conducted according to OECD Test Guideline 210 and in compliance with GLP, a NOEC and LOEC for hatching success, normal larvae at hatch, posthatch survival and growth were concluded to be 0.5 mg/L and >0.50 mg/L, respectively, based on nominal concentrations. The NOEC and LOEC for hatching success, normal larvae at hatch, posthatch survival and growth were concluded to be 0.036 mg/L and >0.036 mg/L respectively, based on geometric mean measured concentrations.

Description of key information

Long-term toxicity to fish: 32-day (28-day post hatch) EC10 >0.5 mg/l and NOEC ≥0.5 mg/l nominal, >0.036 mg/l and ≥0.036 mg/l, respectively geometric mean measured concentration, effects of triethoxy(octyl)silane (CAS 2943-75-1) on hatching success, normal larvae at hatch, post-hatch survival and growth of Pimephales promelas.

Key value for chemical safety assessment

Additional information

Measured long-term toxicity to fish data are available with the registration substance.

A 32-day (28-day post hatch) EC10 value of >0.5 mg/l and NOEC value of ≥0.5 mg/l have been determined for the effects of triethoxy(octyl)silane (CAS 2943-75-1) on hatching success, normal larvae at hatch, post-hatch survival and growth of Pimephales promelas based on nominal concentrations. The EC10 and NOEC values are >0.036 mg/l and ≥0.036 mg/l, respectively, based on geometric mean measured concentrations (Smithers, 2020).

The focus of the chemical safety assessment for this substance is the parent substance, due to its hydrolysis half-life of 30 hours at pH 7. Measures were taken during the long-term toxicity to fish test to maximise exposure to the parent, including use of a solvent for media preparation, keeping the pH neutral and using a semi-static daily renewal system. Although these steps were taken to maximise exposure to the parent substance, measured analytical concentrations of the parent substance showed degradation between test solution renewals.

Analysis of the freshly prepared media at a nominal concentration of 0.50 mg/l showed measured concentrations to range from 67% to 107% of nominal throughout the duration of the test. These results indicated that the test organisms were initially exposed to concentrations close to nominal at each media renewal.

Analysis of the 4-hour old media on Day 0 (test start) showed a measured concentration of 68% of nominal for the 0.50 mg/l test concentration. This decreased to 33% of nominal after the first 24 hours of exposure (Day 1). On Day-0 post-hatch, the measured concentration in the 4-hour old media sample was 36% of nominal which decreased to less than the LOQ after 24-hours (Day-1 post-hatch). For the remainder of the test the 4-hour old sample results ranged from 8% to 24% of nominal with the 24-hour old samples generally less than the LOQ. These results showed the test substance was unstable in the test media but may also interact with biological material as the decrease in measured concentration over the 4- and 24-hour periods increased as the organisms hatched and grew.

A similar trend in analytical measurements was observed for the remaining test concentrations.

Due to the decrease in measured concentration over each 24-hour exposure period, it was considered appropriate to base the results on geometric mean measured.