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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2 October 1990 to 11 February 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Principles of method if other than guideline:
The basic principles behind OECD guideline 210 were followed, however, using different test set-up, test duration, etc.
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Frequent sampling (more or less once a week) for test substance analysis. No data on sampling method in the report itself, but can be assumed to be the same as in Lackner (1991) - see aquatic bioaccumulation.
Vehicle:
no
Details on test solutions:
Every three days, new stock solutions were prepared for use in the peristaltic continuous flow system.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Preore (Italy)
- Freshly fertilised eggs were sampled as well as eggs in the eye dot stage.
- Additionally, 1-yr-old trout were used.

ACCLIMATION
- The 1-yr-old trout were acclimated during only half a week (due to time constraints).

FEEDING
- The test with freshly fertilised eggs was finished as soon as the yolk sac was completely resorbed. In this test fish larvae were not fed.
- In the test with the eggs in the eye dot stage fish were fed as soon as the yolk sac was resorbed, i.e. ca. 3 weeks post-hatch.
- 1-yr-old trout were also fed.
- Type/source of feed: Tagger C19/888 for the 1-yr-old trout, EWOS-Lachsstarter - 0/52 - F for the juveniles. In one experiment a post-experimental phase was added in which live food was supplied (not specified) to check whether the overall condition of the fish would increase.
- Amount given: not reported, but in the test with the eggs in the eye-dot stage, the amount was significantly reduced after the first histological observations to check whether this would improve the overall condition of the fish.
- Frequency of feeding: once daily for the 1-yr-old trout, 4-5 times daily for fish larvae with fully resorbed yolk sac, but frequency reduced to once daily after a while.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
132 d
Remarks on exposure duration:
132 d for the test with 1-yr-old trout, 126 d for the test with the eggs in the eye-dot stage, 56 d for the test with the freshly fertilised eggs
Post exposure observation period:
None, but a post-experiment was performed after the test with the eggs in the eye dot stage with 3 controls only to check if further food reduction would result in further improvements of the overall condition of the fish (2 groups) or whether live food would be even better (1 group).
Hardness:
110 mg CaCO3/L
Test temperature:
October: 12.0°C
November: 10.2°C
December: 7.5°C
January: 7.3°C
pH:
Test with fertilised eggs: 7.91-7.96
Test with 1-yr-old trout: 7.74-7.84
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
Average measured concentration NMMO was 9.28 mg/L
Details on test conditions:
Test with 1-yr-old trout
- 2 aquaria with 30 fish each at the start of testing)
- During this test test substance analysis was performed.
- Fish were used for haematological observations as well as general observations
Test with eggs in eye dot stage
- 2 aquaria, density not reported, density reduced several times in view of improving overall water quality
- Fish were used for histological observations as well as general observations
Test with freshly fertilised eggs
- 2 aquaria, density not reported
- Fish were used for observations of mortality, teratogenicity, and histological observations (the latter only at the end of the test)

General conditions:
- 24-L aquaria were used
- Continuous flow-through (peristaltic) - 270-572 mL/min over all experiments
- For each test two controls were used
- Test medium was tap water, activated carbon filtered:
* pH 8.2
* Conductivity 223 µS
* Alkalinity 1.77 mval
- 12L:12D light cycle
- Aquaria were rinsed twice a day (removal of faeces and part of the water)

Hematological and histological investigations:
- Blood samples were fixed with methanol and stainged using Pappenheim staining method.
- Histological samples were stored in 5% formaldehyde solution. Samples were taken from gills, liver and kidney. After a few days of preservation in the formaldehyde solution samples were dewatered using ethanol-methacrylate solution and fixed in methacrylate (slices of 3 µm thickness). Stainings used: Pappenheim, Hematoxilin-Eosin, PAS.
Reference substance (positive control):
no
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: larval mortality and development
Duration:
126 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: larval growth and mortality
Duration:
132 d
Dose descriptor:
NOEC
Effect conc.:
>= 9.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: mortality, growth, condition factor, liver somatic index
Details on results:
- Larval development:
* No significant effects compared to the control were observed for % normal developing eggs, % larval mortality, and % deformed larvae immediately post-hatch and at the end of the yolk-sac stage. NOEC >= 9.28 mg/L.

- Mortality and growth:
* In the test with the eggs in the eye dot stage no significant effects were observed on growth and mortality compared to the controls. At the end of the test a Costia infection occurred, which is a typical infection for fish with lowered resistance. However, an additional experiment showed that control fish were also succeptible to the Costia infection and that only a minor part of the increased mortality may be attributed to exposure to the test substance.
* In the test with the 1-yr-old trout no significant effects were observed on growth, mortality, condition factor and liver somatic index compared to the controls.

- Hematological observations:
* No significant differences were observed between the different exposed groups and the control.

- Histological observations:
* For NMMO, chlorid cell proliferation in the gills (which assist in sustaining ionic balance) was observed after the first and second sampling. Part of this condition may be attributed to exposure to the test substance.
* For NMMO, thickening of the gill filaments (which results in a decrease of the respiratory surface) was observed at both samplings, although this condition had clearly improved by the second sampling.
* For all treatments including the control, necrosis of liver parenchym was observed (increased level compared to normal), but this was demonstrated to improve when feeding was reduced (and an additional experiment indicated that it improved even more when live food was provided).
* No other significant effects were observed in the liver (mitosis, liver fat, ...).
* For NMMO, no significant increase was observed compared to the control for necrosis of epithelium of tubuli, mitosis, number of pinocytosis vesicles and vacuolar changes in the epithelium of the tubuli, etc.
* An increase compared to the control was observed of hyaline dystrophy of (1st) proximal tubuli. However, this too may have been partly due to the high feeding rate (as demonstrated for the control) and hence only part of this condition may be attributed to exposure to the test substance.
* In all treatments, an increased level of accumulation of glycogen in epithelium of tubuli was observed, indicating that there is a disturbed carbon hydrate metabolism. This may have been linked to the high feeding rates.
* Overall, only minor histological changes were observed, some of which were also noticed in the control and were shown to be linked to the high feeding rate. At the concentration tested NMMO is therefore not considered toxic to early life stages and juvenile rainbow trout. Only part of the observed histological changes may be attributed to NMMO. These changes did not result in macroscopically observable effects.
Reported statistics and error estimates:
Not reported (p level used seems to be 0.01).
Conclusions:
In a limit study using a concentration of 9.28 mg/L N-methylmorpholine oxide no effects were observed that were connected to application of the test substance. Therefore, the NOEC was determined to be >= 9.28 mg/L.
Executive summary:

In this study, several experiments were conducted with freshly fertilised eggs, eggs in the eye dot phase, and 1-yr-old fish (rainbow trout, Oncorhynchus mykiss). In a limit test, a single concentration was tested (9.28 mg/L, mean measured). No effects on development, growth, mortality, etc. were observed in these experiments. No hematological changes were observed either. Some histological changes were observed, but these were only minor. Moreover, some of the changes were also observed in the control and were demonstrated to be linked to the high feeding rate. Improvement of the histological condition was observed when feeding rate was reduced. Consequently, only part of the observed histological condition may be attributed to exposure to the test substance. Further, the histological effects clearly did not result in macroscopically observable effects. The results of this study can be considered reliable but should not be used for PNEC derivation as only one (rather low) concentration was tested and only an unbounded NOEC is available from this study.

The toxicity study is classified as supplementary and satisfies general requirements for early life toxicity study with fish.

Results synopsis

Test organism size/age: freshly fertilised eggs, eggs in the eye dot phase, 1 year old fish

Test type: continous flow through

NOEC: 9.28 mg/L (mean measured)

Endpoint(s) effected: sublethal effects

Endpoint:
fish life cycle toxicity
Data waiving:
other justification
Justification for data waiving:
other:

Description of key information

Based on comparison of the PNECs identified in the hazard assessment with exposure considerations, no hazard was identified (RCR <1) and testing of long term toxicity to fish is not required according to REACH Annex IX, column 2.

Nevertheless, a reliable (Klimisch 2) study is available (Hofer, 1991) and reported as supporting evidence of the absence of aquatic long-term toxicity. However, this study cannot be considered as a key study for the CSA because it used only one exposure concentration at which no effects on development, growth, mortality, etc. were observed. The resulting unbounded NOEC value is therefore not suitable for PNEC derivation.

Key value for chemical safety assessment

Additional information

In the study of Hofer (1991), several experiments were conducted with freshly fertilised eggs, eggs in the eye dot phase, and 1-yr-old fish (rainbow trout, Oncorhynchus mykiss). At the single concentration tested (0.98 mg/L, mean measured), no effects on development, growth, mortality, etc. were observed in these experiments. The results of this study can be considered reliable but should not be used for PNEC derivation as only one (rather low) concentration was tested and only an unbounded NOEC is available from this study.