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Ecotoxicological information

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:
1979-09-28 to 1979-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: ASTM (1979) Standard practice for conducting toxicity tests on the early life stages of fishes. Draft No. 2. US EPA (1972) Proposed recommended bioassay procedures for egg and fry stages of freshwater fishes. Unpublished manuscript. 
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Remarks:
Photoperiod was 16 hours rather than darkness until 1 week after hatching and subdued light thereafter. Water hardness was only determined in the dilution water. Intervals of water quality measurements >1 week.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations tested

- Sampling method: The actual concentrations of test material were determined on days 0, 1, 5, 10, 20, 30, 40, 50, 60 and 66. One tank from each duplicate at each toxicant concentration was analyzed for each sample period.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The stock solutions were prepared on a weight:volume basis by dissolving in deionized water and were delivered to the diluter from a Mariotte bottle enclosed in aluminium foil. New stock solutions were prepared as required. Before initiating the biological portion of the study, the test solutions were allowed to flow through the test aquaria for a 24 hour equilibration period. The test concentrations were confirmed by spectrophotometric analysis before introducing the embryos.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM

- Common name: rainbow trout
 
- Source: The eggs used to initiate this test were obtained from Spring Creek Hatchery, Lewiston, Montana. The eggs were obtained from 3 year old fish. The eggs were held at 10 +/- degree C for 24 hours prior to testing.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS

- Subsequent handling of eggs: All tests eggs were held in a Health Techna vertical incubator cabinet.


POST-HATCH FEEDING
From the latter part of the sac-fry stage and until day 30 of growth, all trout were fed live brine shrimp nauplii in combination with a standard commercial fish food (Rangen's ) 3 t o 4 times a day ad libitum. After growth day 30 and until the termination of the study, the juvenile trout were fed twice daily with Rangen's fish food ad libitum.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
60 d
Post exposure observation period:
no post exposure period
Hardness:
255 ppm as CaCO3 in dilution water
Test temperature:
Temperature maintained at 10 +/- 2 degrees C. 
pH:
The pH of the test media ranged between 7.0 in the high concentration to 8.3 in the low concentration.
Dissolved oxygen:
Lowest value 6.4 mg/L throughout the test (Day 50 in the control). The report states that in one occasion the oxygen saturation fell below 60% however 6.4 mg/L is the lowest value reported and it did not appear to adversely affect the control organisms.
Salinity:
Not Applicable
Nominal and measured concentrations:
Nominal test concentrations were 6, 11, 23, 45 and 90 mg/L (active acid). 
Mean measured concentrations were 4.9, 12.5, 23, 47.6 and 89.4 mg/L (active acid). 
Details on test conditions:
TEST SYSTEM

- Emybro cups (if used, type/material, size, fill volume): cups suspended in the vessels. Egg incubation cups were made from 7.0 cm diameter polyethylene tubing with stainless steel screening (16 mesh) welded to the bottom.

- Test vessel:

- Material, size, headspace, fill volume: glass aquaria measured 36x30x30 cm with water depth of 24 cm. Each growth aquaria was divided by a glass partition to provide space for 2 growth chambers measuring 25x15x30 cm and had a stainless steel screening attached to one end.

- Aeration: aerated well water was delivered to the vessel

- Type of flow-through (e.g. peristaltic or proportional diluter): Mount & Brung proportional diluter. 

- Renewal rate of test solution (frequency/flow rate): replacement rate of 100 ml/min/test vessel. 

- No. of fertilized eggs/embryos per vessel: Test initiated with a total of 200 eggs per concentration. After hatching, the number of fry was reduced to 4 groups of 20 per concentration. 

- No. of vessels per concentration (replicates): 1

- No. of vessels per control (replicates): two replicates with eggs, then 4 with fry.


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: ABC Aquatic Bioassay Laboratory's well water

- Metals:<0.01 ppm

- Pesticides: <110 ng/L

- Alkalinity: 368 ppm as CaCO3

- Conductivity: 50 µmho/cm

- Culture medium different from test medium: no

- Intervals of water quality measurement: Temperature, DO, and ammonia were measured on days 0, 4, 7, 20, 30, 40, 50, 60 and 66 in control, low concentration, and high concentration samples


OTHER TEST CONDITIONS

- Photoperiod: 16h daylight

- Light intensity: eggs shielded from UV exposure.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): the effects of test material were determined on hatchability, survival, growth, behaviour and morphological changes of the embryos and fry, and recorded at least weekly. The eggs were inspected daily and dead eggs removed. Growth as determined by standard length of the fry was determined by the photographic method of McKim and Benoit (13) immediately after transfer of the fry to the respective growth chambers and at 15, 30, 45 and 60 days thereafter.




POST-HATCH DETAILS

- Begin of post-hatch period: since embryo hatching was spread out over 10 days , the modal hatch date was used to establish day 0 for growth sampling periods.

- No. of hatched eggs (alevins)/treatment released to the test chamber: 20 per chamber
Reference substance (positive control):
no
Duration:
60 d
Dose descriptor:
NOEC
Effect conc.:
23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: length and weight of fry
Duration:
60 d
Dose descriptor:
LOEC
Effect conc.:
47.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: length and weight of fry
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: The percentage survival of the fry when continuously exposed to the test material for 60 days was not significantly affected. See Table 1 for details.

- Numbers hatched:: Hatchability of rainbow trout eggs continuously exposed to the test material was not significantly (P=0.05) reduced at any concentration compared to the control eggs. See Table 1 for details.

- Observations on body length and weight of young and/or exposed parents at one or more time periods: Growth of the fry, as measured by length, was significantly reduced (P=0.05) after 45 and 60 days of exposure to 47.6 mg/L and 89.4 mg/L test material. Wet weights of the rainbow trout fry were also significantly reduced after 60 days exposure to these concentrations. See Table 1 for details.

- Other biological observations: Observations also indicated that the trout fry at concentrations 47.6 and 89.4 mg/L exhibited a noticeably excitable behaviour. Eggs at 89.4 mg/L had a whitish colour or coating prior to hatching.
Reported statistics and error estimates:
Hatching, survival and growth data was subject to analysis of variance.

Table 1. Mean hatch, survival and growth (length and weight).

 

 Mean measured conc. (mg/L)

 

1 -15 days

16 -30 days

31 -45 days

46 -60 days

 

 

  Mean Hatch (%)

Survival (%) 

 Mean Length (mm)

 Survival (%)

  Mean Length (mm)

 Survival (%) 

  Mean Length (mm)

 Survival (%) 

  Mean Length (mm)

 Mean Tot. wet weight (g)

 Control

 97

 100 

20 ± 1.6

71

27 ± 2.5

70

32 ± 3.4

70 

40 ± 4.5 

1.1 ± 0.36 

 4.9

 95

 98

21 ± 1.5* 

86

27 ± 2.2 

86 

32 ± 2.4

86

40 ± 2.9 

1.1 ± 0.28 

 12.5

 97 

 98

20 ± 1.6

84

27 ± 2.2 

83 

32 ± 3.2

84 

39 ± 4.2 

1.0 ± 0.39 

 23

 95

 96 

21 ± 1.6* 

89

27 ± 2.5 

88 

32 ± 3.1

88 

39 ± 3.9 

1.0 ± 0.31 

 47.6

 97

 98 

20 ± 1.5

85

26 ± 2.4

83 

29  ± 3.4*

81 

37 ± 4.3* 

0.83  ± 0.28*

 89.4

 97

 98

20 ± 1.5

86

25 ± 2.2* 

83 

26 ± 2.6*

 78

30 ± 4.0* 

0.45 ± 0.19* 

* denotes a statistically significant difference from the control group (p = 0.05).

Table 2.Nominal and measured concentrations during 66 days

 

Measured concentration (Unit) results

Nominal conc. (mg/L)

0 (control)

6

11

23

45

90

Range (min.-max.)

 0-0.48

3-7 

11-14 

19-27

40-56

 71-105

Mean ± st. dev.

0.19

4.9 ±1.5

12.5 ± 0.72 

23 ± 2.3

47.6 ± 5.51

89.4 ± 9.6 

% of nominal (ref. to mean)

80% 

113% 

100%

106% 

99.5% 

 

Validity criteria fulfilled:
no
Remarks:
Whilst the validity criteria for the study were not met (D.O. <60%) saturation this did not adversely affect the outcome of the study.
Conclusions:
In a reliable 60 day ELS study a 60 d NOEC value of 23 mg/L (as active acid) has been determined for the effects of the test substance on growth of the fish O. mykiss.

Description of key information

(60 d) NOEC 23 mg (active acid)/L

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
23 mg/L

Additional information

A 60-day NOEC value of 23 mg/L (as active acid) has been determined for the effects of ATMP-H on growth of the fish O. mykiss. This study represents the only value available for this endpoint.