4-methylmorpholine

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish, juvenile growth test

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990-10-02 - 1991-02-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study but non-guideline and non-GLP. Only a single concentration was tested, resulting in unbounded NOEC values. However, the results of the study can be considered reliable (Klimisch 2).

Qualifier:

equivalent or similar to guideline

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 NMM was 0.98 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

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.98 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: larval mortality and development

Key result

Duration:

126 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.98 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: larval growth and mortality

Key result

Duration:

132 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.98 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 >= 0.98 mg/L.

- Mortality and growth:
* In the test with the freshly fertilised eggs at the end of the larval stage there was a slight but significant effect on growth (p < 0.01).
* In the test with the eggs in the eye dot stage no significant effects were observed on growth and mortality compared to the controls.
* 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 NMM, chlorid cell proliferation in the gills (which assist in sustaining ionic balance) was observed after the first sampling only. Afterwards this condition returned back to normal.
* For NMM, 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 NMM, no significant increase was observed compared to the control for necrosis of epithelium of tubuli, hyaline dystrophy of (1st) proximal tubuli, mitosis, ...
* 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.
* For NMM, an increase in the number of pinocytosis vesicles and vacuolar changes in the epithelium of the tubuli was observed at the second sampling.
* 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 NMM is therefore not considered toxic to early life stages and juvenile rainbow trout.

Reported statistics and error estimates:

Not reported (p level used seems to be 0.01).

Conclusions:

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). At the single concentration tested (0.98 mg/L, mean measured), no effects on development, growth, mortality, etc. were observed in these experiments. No hematological changes were observed either. For NMM, 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. 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.

Description of key information

One reliable (Klimisch 2) study was available (Hofer, 1991). 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.

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.