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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:
weight of evidence
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited information on test material and no information on GLP. Peer reviewed article.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The toxicity of the test material was investigated in early life stages of the rainbow trout. Beginning with yolk sac fry, trout were continuously exposed for 110 days to the test material at concentrations of 0.12-15 nM. Histopathological examinations were carried out at the end of the study and an NOEC was determined.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
yes
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Eyed rainbow trout eggs were obtained from Jorgenson, Ny Molle Dambrug Trout Hatcheries, Denmark, and from Clearwater Trout Ltd, Kirk Michael, Isle of Man, Great Britain.
They were kept in the laboratory in flowing municipal tap water (temperature, 11 ± 2 °C; hardness, 96-105 ppm CaCO3; pH, 7.35-7.45). After hatching, the yolk sac fry were placed in 8-litre glass aquaria through which water continuously flowed at a rate of 4 L h^-1, containing oxygen at about 90-100 % saturation. Aquaria were continuously aerated, and maintained on a 16 h light/8 h dark photoperiod. Aquaria were cleaned two times a week.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
110 d
Details on test conditions:
Trial I was carried out in summer. Each aquarium contained 100 fry at the beginning of the trial and the water temperature was 14 ± 2 °C. Trial II was carried out in the winter period. The management was identical as in Trial I except that each aquarium contained 50 fry at the beginning of the trial. The water temperature was 13 ± 2 °C.
From the moment the yolk sac fry started swimming (about the tenth day after hatching), they were fed a commercial ration (Trouvit, Trouw and Co N.V., Putten, the Netherlands). The dry feed was given five times daily on working days and two times daily during weekends. The daily amount was ~3-5 % of the body weight. Mortality and abnormal behavioral symptoms were recorded daily.
The test material was investigated at concentrations ranging from 0.12 to 15 nM. The fish were exposed for 110 days in a continuous dosing system.
Test material stock solutions were prepared in acetone and continuously mixed with tap water to obtain the target concentrations using an automatic dosing apparatus (Perfusor Secura E, B. Braun, Germany). The final acetone concentration in water was 225 ppm (v/v). Controls were exposed to 225 ppm (v/v) acetone. Stock solutions were freshly prepared twice a week.
Total body lengths of eight fry from each aquarium in Trial II were measured on Day 11 of exposure. Individual body weights of 15 specimens from each aquarium were recorded at weekly intervals from Week 7 of exposure.

Histopathology:
In those exposure groups in which mortality occurred within a few days of exposure, five fish per group were sampled and processed for histological examination. Fish were anaesthetised in buffered tricaine methane sulfonate (MS222, Sandoz, Basel, Switzerland), fixed in Bouin for 24 h, dehydrated and embedded in paraffin wax. Transverse serial sections of 5 µm thickness were made at 100 or 200 µm intervals and stained with haematoxylin, phloxin and saffran (HPS) and/or periodic acid-Schiff (PAS). The amount of glycogen in the liver was judged semi-quantitatively by estimating the relative number of vacuoles in the liver sections. After 1 month of exposure, five fish were taken from each aquarium for whole body histopatholgy, including semi-quantitative determination of liver glycogen content.
After 110 days of exposure, 10 fish from each group that survived the treatment were killed and necropsied and body and liver weights were recorded. Several organs (liver, thymus and spleen) were sectioned and stained with HPS and/or PAS. The caudal part of the spinal cord of three fish with black tails was also processed for microscopic examination (HPS).
For thymus morphometrical analysis, transverese sections were made at 100 µm intervals. The circumference of each section was drawn, with the help of a drawing objective on the microscope, and measured.
Duration:
110 d
Dose descriptor:
NOEC
Effect conc.:
0.12 other: nM
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
MORTALITY
The test material caused a dose-related mortality. All fry exposed to 15 nM of the test material died within the first 14 days of exposure. At 0.12 nM no increased mortality was observed.

BEHAVIOURAL EFFECTS
Fry exposed to the highest sub-lethal concentration of the test material (0.12 nM) were generally lethargic and usually swam in the lower half of the aquarium.

TAIL MELANISATION
Fish with black tails were also observed at 3 nM.

BODY WEIGHTS
Body weights of young rainbow trout were recorded at weekly intervals from Week 7 of exposure. Significantly decreased body weights were observed in all groups exposed to levels of the test material which caused increased mortality rates. In groups in which little or minimal mortality occurred, body weights of the exposed animals were in the same range as those of the controls. The highest exposure levels of the test material that did not affect body weight was 0.6 nM.


ORGAN WEIGHTS
The hepatosomatic index was significantly increased after exposure to 0.6 and 3 nM test material. Mortality also occurred at these concentrations. At lower concentrations no effects were observed. Spleen weights were not affected.

HISTOPATHOLOGY
Histopathological examination detected no morphological changes in gills, heart, intestine, skin, kidney, spleen, thymus and brain at various time intervals during the 110 days exposure to the test material. Only liver glycogen depletion was found at various concentrations of the test material. After 1 month exposure, liver glycogen depletion was observed at 3 nM. After 110 days exposure, the glycogen content of liver sections from 0.6 and 3 nM exposed fish was decreased. The black tails; occurring in some of the exposed fish, were probably caused by dispersion of melanin pigment. No abnormalities were found in the spinal cord and no signs of dermatitis were observed in the skin of these animals. After 1 and 3 months exposure to 3 nM test material, the highest sub-lethal concentration, no atrophy or other changes were observed in the thymus.

RESISITANCE TO BACTERIAL CHALLENGE
At concentrations of 0.6 nM test material a significantly decreased resistance against challenge with A. hydrophila was found.


A NOEC of 0.12 nM was established for the test material, corresponding to 0.039 µg/L.
Reported statistics and error estimates:
For comparing means, analysis of variance and subsequent t-test were used according to the least significant difference test proposed by Fisher. For comparing frequencies or proportions, Fisher's exact test was used.
Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of this study, a NOEC of 0.12 nM was established for the test material, corresponding to 0.039 µg/L
Executive summary:

The toxicity of the test material was investigated in early life stages of the rainbow trout.

Beginning with yolk sac fry, trout were continuously exposed for 110 days to the test material at concentrations of 0.12-15 nM. The test material caused acute mortality at a concentration of 15 nM. A NOEC of 0.12 nM was established, corresponding to a water concentration of 0.039 µg/L.

Histopathological examination revealed depletion of glycogen in liver cells of the test material exposed fish. Atrophy of the thymus, the most prominent sign of toxicity of di- and tributyltin compounds in mammalian species, was not observed in early life stages of rainbow trout. Tail melanisation was observed in the groups exposed to the test material at 3 nM.

At the end of the exposure period, resistance to infection was examined by an intraperitoneal challenge with Aeromonas hydrophila, a secondary pathogenic bacterium to fish. Resistance of bacterial challenge was found to be decreased even at the lowest-effect concentration of triorganotin compounds.

Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Purity > 98 %. Study unlikely to be GLP. Details on method used documented, will not be according to OECD method but study seems well performed.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rainbow trout in the yolk sac fry stage were continuously exposed to the test material at concentrations of 0, 0.2, 1 and 5 ppb (approximately equivalent to g/L) for 110 days. Clinical abnormalities and mortality were recorded during the whole experimental period. After 110 days, 15 fish from each group that survived the experimental period were stunned and necropsy was performed, along with histopathological and haematological exminations.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
Stock solutions were prepared in absolute ethanol and continuously mixed with tap water (pH 6.8, hardness 94 - 102 ppm CaCO3), at concentration of 0, 0.2, 1 and 5 ppb (µg/L) by means of an automatic dosing apparatus. Final ethanol concentration in water was 0.001 %. Stock solutions were freshly prepared twice a week.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Freshly fertilised eggs were obtained from the Dutch Trout Hatcheries and bred in the laboratory in flowing tap water at 10 °C. After hatching, the yolk sac fry were placed in 8 L glass tanks through which tap water continuously flowed at a rate of 16 L/h at a temperature of 14 ± 1 °C and contained 9 ± 1 ppm O2 during the experimental period for 110 days. From the transition to the stage of swimming fry all animals were fed a commercial ration (Trouvit, Trouw and Co N.V., Putten, The Netherlands) 3 times daily.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
110 d
Nominal and measured concentrations:
Nominal: 0, 0.2, 1 or 5 ppb (µg/L)
Details on test conditions:
- Six-hundred yolk sac fry were equally divided into 4 groups and exposed to concentrations of 0, 0.2, 1 or 5 ppb test material. Each group was kept in 3 aquaria each containing 50 fish. Individual body weights for ten fish from each aquarium were recorded at weekly intervals from day 55. Clinical abnormalities and mortality were recorded during the whole experimental period.
- After ten days of treatment, when mortality occurred in the 5 ppb group, ten fish from each group were sampled and processed for histopathological examination. These early swimming fry stage fish were anaesthetised in buffered tricaine methane sulfonate (MS222, Sandor, Basel, Switzerland) fixed in Bouin Hollande for 24 h, dehydrated and embedded in Paraplast.
- Transverse serial sections of 7 µm thickness were made at 100 µm intervals and were stained with haematoxylin and eosin.
- After 110 days, 15 fish from each group that survived the experimental period were stunned and necropsy was performed. Body and liver weights were recorded. Samples of gills, heart, liver, brain, thymus (of 5 fishes only), kidneys, spleen, skin and muscle were fixed in Bouin Hollande and processed for histopathological examination by conventional means. Sections were stained with haematoxylin and eosin. Liver sections were analysed morphometrically by the method of Weibel using a 25-points integrating eyepiece for the determination of the relative area of nucleus, glycogen storage, other cytoplasmic components and non-parenchymal tissue. In liver sections of 10 fish from each group point counts were made of 15 random eye fields at a 250 x magnification.
- From ten animals of each group, the thymus was trimmed free from the adjoining tissue (under a stereomicroscope) and sampled in cold phosphate buffered saline at ph 7.4 and an osmolarity of 220 m Osm. Thymus tissue was minced with scissors into fragments which were gently pressed through a nylon sieve with a pore diameter of 220 µm. The cell viability was determined by trypan blue exclusion and the number of cells present was determined with an electronic particle counter (Coulter counter, model ZF, Coulter Electronics, Dunstable, Bedfordshire, England).
- For the haematologic investigations blood was drawn from the heart using heparinised capillaries. The haemoglobin content was determined spectrophotometrically by a microassay using 5 µL of blood. Blood cells were counted with an electronic particle counter.
Reference substance (positive control):
not specified
Duration:
110 d
Dose descriptor:
NOEC
Effect conc.:
< 0.2 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
MORTALITY
After the transition of the yolk sac fry stage to the stage of swimming fry, which occurred from day 10-12 of treatment all animals of the 5-ppb group died. In the other test groups, mortality was within the normal range for the early-life stages of trout and was comparable to the mortality in the control group (Table 1).

GROWTH
A dose-related growth retardation was observed in both test groups that survived the experimental period (Fig. 1). Body weights for fish of the 1-ppb group were significantly less than the controls from the first time of measurement (day 55) and at the end of the experimental period the mean body weights were only 56 % of the controls. At the 0.2 ppb level growth retardation was less pronounced. Body weights were significantly lower than the controls only during the last weeks of treatment.

HAEMATOLOGY
The haemoglobin concentration of fish exposed to the 0, 0.2 or 1 ppb concentration for 110 days (Table 2) indicated a statistically significant and dose-related decrease. The number of blood cells was significantly decreased at the 1-ppb level only.

ORGAN CHANGES
At microscopic examination treatment-related pathological changes were noted in kidney and liver. In fish, exposed for 10 days to the lethal dose of 5 ppb TBTC, extensive hydropic degeneration of tubule segments of the pronephros was observed. The liver of these fish was almost completely depleted of glycogen. At lower exposure levels no histological changes were noted after 10 days exposure.
In fish exposed for 110 days the relative liver weights were dose-relatedly increased (Table 3). According to these weight changes histopathological alterations were noted both at the 0.2 and 1 ppb dose levels. In these fish hyperplasia of liver cells and a diminished storage of glycogen was noted. At the 1 ppb test material level the nuclei of hepatocytes were arranged in rosettes. Their cytoplasm was swollen and vacuolated.
Quantification of these changes by morphometric analysis showed that the total area of liver nuclei was dose-relatedly increased, whereas the area of glycogen storage was dose-relatedly decreased (Table 4). The other cytoplasmic and non-parenchymal compartments such as vessels, sinusoids, bile ducts and stromal fibres were equally present in liver sections of test and control fish. Since no enlargement of liver cells was established and multinuclear cells were not prominent, the increase on nuclear area has to be ascribed to an increase in the number of liver cells. In contrast to the highest (lethal) exposure level, no kidney changes were observed. Also in the other organs examined, no treatment-related histopathological changes were observed. In relation to the thymolytic effect of the test material in rats the relative number of cells isolated from the thymuses of young trout was determined, but this was not affected.
Reported statistics and error estimates:
The data were analysed for significance of differences by Student's t-test.
Validity criteria fulfilled:
not specified
Conclusions:
This study reveals that a test material concentration of 5 ppb was lethal for the early life stages of rainbow trout and the "no-toxic-level" is < 0.2/µg/L.
Executive summary:

Rainbow trout in the yolk sac fry stage were continuously exposed to the test material at concentrations of 0, 0.2, 1 and 5 ppb (µg/L) for 110 days.

After a 10 to 12 day exposure period, at the transition of the yolk sac fry stage to the swimming fry stage, all fish of the 5 ppb group suddenly died without any preceding symptoms.

Hydropic degeneration of tubule segments of the pronephros was the main finding in these animals, whereas no histopathological changes were noted in animals exposed to 0.2 or 1 ppb for 10 days. At these exposure levels the test material induced a significant and dose related growth retardation resulting in a 44 % decrease of the body weights in the 1 ppb group at the end of the experimental period. Haemoglobin content of the blood was decreased in a dose-related fashion and the number of red blood cells was significantly decreased at the 1 ppb exposure level. Histopathologically hyperplasia of liver cells was observed and as a consequence, the relative liver weights were significantly increased in a dose-related manner (both at a concentration of 0.2 and 1 ppb).

This study reveals that a test material concentration of 5 ppb was lethal for the early life stages of rainbow trout and the "no-toxic-level" is < 0.2/µg/L.

Description of key information

Key value for chemical safety assessment

Additional information

Weight of evidence information is available for this endpoint:

- In the De vries et al, 1991 paper, the toxicity of the test material was investigated in early life stages of the rainbow trout. Beginning with yolk sac fry, trout were continuously exposed for 110 days to the test material at concentrations of 0.12-15 nM. The test material caused acute mortality at a concentration of 15 nM. For the test material a NOEC of 0.12 nM was established, corresponding to a water concentration of 0.039 µg/L.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although this is a non-standard study with no information on GLP the methods are well documented.

- In the Seinen et al, 1981 paper, Rainbow trout in the yolk sac fry stage were continuously exposed to the test material at concentrations of 0, 0.2, 1 and 5 ppb (µg/L) for 110 days. After a 10 to 12 day exposure period, at the transition of the yolk sac fry stage to the swimming fry stage, all fish of the 5 ppb group suddenly died without any preceding symptoms. This study reveals that a concentration of 5 ppb test material was lethal for the early life stages of rainbow trout and the "no-toxic-level" is < 0.2/µg/L.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as although this is a non-standard study with no information on GLP the methods are well documented.