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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:
10 July - 18 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD guidelines and in compliance with GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from both test groups. Samples were taken in duplicate from freshly prepared solutions, while singular samples were taken from each of the spent solutions per replicate. The filter containing the undissolved test substance residue was kept for possible analysis.
Frequency: At the beginning and at the end of two intervals of 72 hours (nominal days 16 and 19, 30 and 33) and five intervals of 48 hours (days 0 and 2, 7 and 9, 14 and 16, 21 and 23, 28 and 30).
Volume: 2.0 mL from the total solution freshly prepared and from the old solutions of each replicate vessel.
Storage: Samples not analysed on the day of sampling were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
Test solutions were prepared at the limit of solubility in test medium by magnetic stirring of a loading rate of 10 mg/L for three subsequent days. The major fraction of larger undissolved test material was then removed by filtration through a rough paper filter (Whatman 597; pore size ~4-7 µm, except day 14 and 16: Whatman 604½; pore size ~12-15 µm). The resulting clear and colourless WSF (Water Soluble Fraction) was used for testing. Test solutions were renewed three times a week. The preparation method was based on the method used for the acute toxicity testing performed with SPS-100 in 2001 at NOTOX (Daphnia magna acute toxicity test, NOTOX Project 307518 and algae growth inhibition, NOTOX Project 307529) and the daphnia reproduction test performed in 2012 at WIL Research Europe (project 498776).

Note that the use of a rough paper filter with a relatively high pore size was thought to result in maintenance of actual concentrations slightly above the actual limit of solubility. As such it should confirm exposure and thus prevent that test concentrations would all be below the Limit Of Detection (LOD) of the analytical equipment.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas, Teleostei Cyprinidae, Rafinesque
- Source: In house culture

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: ratio male:female is 1:2; Males and females are put together in spawning tanks and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.
- Method of collection of fertilised eggs: The spawning tank is equipped with a substrate (pvc-tube), which enables collection of the fertilised eggs.
- Subsequent handling of eggs: Introduction of the eggs is before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation).

POST-HATCH FEEDING
Embryonic phase: no feeding.
Newly hatched larvae: Brachionus suspensions.
Juvenile stage: Brine shrimp Nauplii 24 or 48-hours old.
Food is supplied ad libitum.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d
Remarks on exposure duration:
28 d post-hatch
Hardness:
196-214 mg CaCO3 per liter
Test temperature:
23.2 -25.7 ºC
pH:
7.3 - 8.3
Dissolved oxygen:
4.9-9.1 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
A WSF prepared at a loading rate of 10 mg/L was tested.
The results of analyses of all samples taken during the 33-day test period indicated exposure to (over)saturated concentrations during the early and most sensitive life stages of the fathead minnow (embryonic and early larval stage) and exposure to at least the maximum soluble fraction in test medium during the later larval stages. Analyses showed that measured concentrations in the WSF were variable and ranged between 6.7 µg/L and 178 µg/L for the first 9 days and ranged between
Details on test conditions:
TEST SYSTEM
- Test vessel: Embryonic phase: Petri-dishes. Early larval phase: stainless steel vessels (1.7 L), late larval stages: all-glass vessels (5 and 8 L).
- Aeration: no
- Renewal rate of test solution: Three times a week (on Monday, Wednesday and Friday). The last renewal was performed on day 30.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photo-period daily
- Light intensity: between 512 and 704 lux

EFFECT PARAMETERS MEASURED:
Organisms
- Stage of embryonic development: Daily, from the beginning of the exposure
- Hatching and survival: Daily from nominal day 3, numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded. Dead embryos, larvae and juvenile fish were removed directly after recording.
- Criteria for death
Eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance;
Embryos: Absence of body movement and /or absence of heartbeat;
Larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus.
- Abnormal appearance: Daily, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and typical swimming behaviour.
- Body weight and length: At the end of the test, all surviving fish were individually weighed and measured.
Test media
- Oxygen, pH and temperature: At the start and the end of the test and at each renewal in all test concentrations. In the freshly prepared solutions measurements were performed before exposing the organisms and in the old solutions after pooling of the replicates per group. In addition, temperature was recorded continuously.
- Hardness: During the first and last renewal interval in fresh and old media from both test groups
- Total Organic Carbon (TOC): At the start of the test in blank-medium

POST-HATCH DETAILS
- No. of hatched eggs (alevins)/treatment released to the test chamber: 80
The experiment (nominal day 0) started with 80 fresh and healthy fertilised fathead minnow eggs per test group. The fertilised eggs were randomly distributed and divided equally over four petri-dishes. Each petri-dish contained 20 eggs in 50 mL test medium and these dishes were incubated under gentle continuous shaking. On day 5 the hatched larvae were transferred to stainless steel test vessels.

Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOEC
Conc. based on:
test mat.
Basis for effect:
other: embryo development, number hatched, time to hatch and larval development
Remarks on result:
other: Since no effects were observed in a WSF prepared at a loading rate of 10 mg/L, the NOEC is considered to be equal to the maximum soluble test substance concentration in test medium.
Details on results:
Analysis was performed on freshly prepared WSF’s and on 48- or 72-hour old solutions. The results showed that it was possible to detect the test substance during the first 9 days of exposure at concentrations clearly above the limit of detection (LOD). From day 14 onwards concentrations measured were generally below the LOD with some excursions just above the LOD mainly in spent solutions (e.g. on days 16 and 19). The LOD was either 2.3 or 2.8 µg/L (depending on the day of analyses). Analyses were also performed on the filter residues that were retained after filtration in cases with measured concentrations below the LOD in the freshly prepared WSF. These results all indicated that the filter residue was in fact SPS-100 and as such confirmed the extremely limited or even insoluble character of this test substance. The insoluble character was already known before initiation of the present Early Life Stage test. A water solubility experiment performed by NOTOX (Project 307362, Brekelmans, 2001) showed that none of the three main components could be detected (all < LOD). As a consequence it was also decided to use a rough paper filter of ~5-10 µm instead of the standard applied 0.45 µm filters to enable detection of at least some SPS-100 in the samples during the present study.
A possible reason for the fact that SPS-100 was detected in relatively high concentrations during the first two renewal periods with analytical support is the relatively low volume in relation to the much higher and increasing volumes needed during the course of the study. All solutions were prepared applying similar stirring time (3 days) and intensity but different volumes. Hence, energy input was likely higher at the study start when volumes were 0.5 L or 5 L, e.g. for days 0 and 7, respectively. The volume increased to 15, 25 and 35 L from days 12, 16 and 26, respectively. Consequently, it can be expected that the test substance was more finely dispersed and therefore capable of passing the filter paper. However, this solution should thus be considered as still supersaturated.

Survival of eggs in the control group was very high at 98%. Survival in the SPS-100 test group was also high at 90%. Statistical analyses showed that there was a difference between survival in the control group and the SPS-100 treatment group. This difference was however considered as biologically irrelevant.
A total of two eggs died in the control while eight eggs died in the WSF all on the 1st day of exposure. Hence, relative dead percentages were 2.5 and 10% for the control and the WSF, respectively. The dead eggs were likely related to some eggs with poor quality that were initially introduced. However, this cannot be confirmed with evidence. It can however be concluded that the lower survival in the WSF is of no biological relevance as it did not exceed 10%. This could be strengthened by the fact that a survival percentage of 90% is very high and often not even achieved for the control. Data obtained during the last 2 years for ELS testing within WIL Research preceding the present test showed a mean embryonic control survival of 88% (n=15, SD=8.7). The embryonic survival of 90% as obtained in the WSF during the present study exceeded the mean survival obtained for the control during the last 15 ELS studies performed within WIL Research. Finally, a background mortality of 30% is allowed for embryo control mortality (validity criterion 70% survival). This once more confirms that 10% mortality should be considered as biologically irrelevant.
Alternatively, an EC10 instead of a NOEC might be applied as endpoint for embryonic survival. The EC10, applied as a worst-case scenario, equalled the maximum solubility in test medium.

No differences were observed in the development of the embryos until hatching in either the control or the treatment group. At the time points of recording, all embryos appeared to be at the same stage of development. It could be concluded that SPS-100 did not affect embryonic survival or development up to and including its maximum solubility in test medium.

The data of hatching starting on the third day and monitored until day five showed that first embryos were in general observed to have hatched in the morning of the third exposure day. On day five all embryos had hatched in both test groups. There appeared to be no difference in time until hatching between the control group and the SPS-100 WSF. It was concluded that SPS-100 did not affect time of hatching or the hatching success up to and including its maximum solubility in test medium.

High larval survival rates were recorded in the controls and the SPS-100 treatment during the whole test period. Survival in the control was 95% and the survival in the WSF was even higher at 97%. Statistical analyses confirmed that there was no difference between survival in the control group and the SPS-100 treatment group.
Except for some minor incidental cases, no visible sub lethal effects in the larvae were recorded in the SPS-100 treatment. There were no statistically significant differences for both body weight and length between the control group and the SPS-100 treatment.


Reported statistics and error estimates:
The following statistical procedures were used to determine the NOEC for embryonic and larval survival, body length and weight.

NOEC for embryonic and larval survival:

• Differences between the treatment and the control: Chi²-2 x 2 table test with Bonferroni Correction (α=0.05, one-sided greater).

NOEC for body length and body weight:

• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• Differences between treatment and the control: Student-t test for homogeneous variances (α=0.05, one-sided smaller).

All analyses were performed with ToxRat Professional 2.10.05 (ToxRat Solutions® GmbH, Germany).

Analytical results of the samples taken during the ELS test

Test group

Measured concentrations (µg/l)

Day 0

fresh

Day 2

old

Day 7

fresh

Day 9

old

Days

14,16,21,28,30

fresh

Days

16,19,23,30,33

old

WSF

74.1-74.9

6.7-8.5

177-178

51.4-72.0

<LOD

<LOD – 29.1

Validity criteria fulfilled:
yes
Conclusions:
SPS-100 did not induce any significant, visible effects on the development of fathead minnow embryos at its maximum solubility in test medium. The test substance did not affect time of hatching or the hatching success nor survival, growth or development of the larvae during the post-hatch period at its maximum solubility in test medium. Hence, the NOEC of SPS-100 for the early life stages of fish equals the maximum soluble concentration in test medium.
Executive summary:

A fish, early-life stage toxicity test was performed with SPS-100 in order to assess its possible lethal and sub-lethal effects during the embryonic and early larval development of the fathead minnow. The study was conducted in accordance with OECD 210 and in compliance with GLP. A WSF was prepared by stirring SPS-100 at 10 mg/L for 3 days and filtration through rough filter paper. Analytical measurements showed that concentrations in the WSF were variable and ranged between 6.7 and 178 µg/L for day 0 until day 9 (the embryonic and early larval stage which are the most sensitive life stages of the fathead minnow) and ranged between < LOD ( < 2.3 µg/L) and 29.1 µg/L from day 14 until day 33 (later larval stages). SPS-100 did not induce any significant, visible effects on the development of fathead minnow embryos at its maximum solubility in test medium. The test substance did not affect time of hatching or the hatching success nor survival, growth or development of the larvae during the post-hatch period at its maximum solubility in test medium. Hence, the NOEC of SPS-100 for the early life stages of fish equals the maximum soluble concentration in test medium.The study was reliable without restrictions.

Description of key information

SPS-100 did not induce any significant, visible effects on the development of fathead minnow embryos at its maximum solubility in test medium. The test substance did not affect time of hatching or the hatching success nor survival, growth or development of the larvae during the post-hatch period at its maximum solubility in test medium.
Hence, the NOEC of SPS-100 (SPB-100, SPE-100) for the early life stages of fish equals the maximum soluble concentration in test medium.

Key value for chemical safety assessment

Additional information

A fish, early-life stage toxicity test was performed with SPS-100 in order to assess its possible lethal and sub-lethal effects during the embryonic and early larval development of the fathead minnow. The study was conducted in accordance with OECD 210 and in compliance with GLP. A WSF was prepared by stirring SPS-100 at 10 mg/L for 3 days and filtration through rough filter paper. Analytical measurements showed that concentrations in the WSF were variable and ranged between 6.7 and 178 µg/L for day 0 until day 9 (the embryonic and early larval stage which are the most sensitive life stages of the fathead minnow) and ranged between < LOD ( < 2.3 µg/L) and 29.1 µg/L from day 14 until day 33 (later larval stages). SPS-100 did not induce any significant, visible effects on the development of fathead minnow embryos at its maximum solubility in test medium. The test substance did not affect time of hatching or the hatching success nor survival, growth or development of the larvae during the post-hatch period at its maximum solubility in test medium. Hence, the NOEC of SPS-100 for the early life stages of fish equals the maximum soluble concentration in test medium. The study was reliable without restrictions.