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Long-term toxicity to fish

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Reference
Endpoint:
long-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The reaction mass of sulphuric acid, hydrogen peroxide and peroxomonosulphuric acid is predominantly sulphuric acid (>80%). Although all constituents of the reaction mass contribute towards and are essential for the desired technical effects of the range, it is considered acceptable to read-across to data on sulphuric acid. This because significant toxicological effects are likely to be masked in the multi-constituent substance by its corrosive nature and so it considered appropriate to read across to the mean constituent, sulphuric acid, when considering aquatic toxicity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across data matrix under 'Attached background material' below.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across data matrix under 'Attached background material' below.

4. DATA MATRIX
See read-across data matrix under 'Attached background material' below.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Effects of low pH on reproduction, growth and survival were investigated in Jordanella floridae
GLP compliance:
no
Remarks:
study pred-dates GLP
Analytical monitoring:
yes
Details on sampling:
- Water samples were collected from the exposure tanks for analysis of heavy metals and water quality parameters.
Vehicle:
no
Test organisms (species):
Jordanella floridae
Details on test organisms:
- Mature flagfish less than one year old were purchased from a local pet supplier and held in the Toronto laboratory dechlorinated water (130 mg/L CaCO3 hardness) for two months before being transferred to the Lake Panache site.
- Male and female flagfish were separated and held in lake water for 7 d in different holding tanks.
- Five females and two males were then randomly selected and placed in each exposure tank.
- Untreated lake water was allowed to flow through all the tanks for an additional 10 d.
- During the following 5 d the pH was linearly depressed to the levels required for the experimental period.
- The control tanks were fed lake water through a diluter channel isolated from the acid delivery system.
- Fish were fed ad libitum on chopped rinsed earthworms and frozen brine shrimp (Artemia sp).
- Debris was vacuumed from the tanks daily after spawning mats were removed for inspection.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
65 d
Post exposure observation period:
- A supplemental experiment was carried out to determine the reproductive response of flagfish to acute pH depression and the extent of any latent effects once conditions returned to normal.
- Duplicate breeding communities were established as for the main test and allowed to spawn for 16 d in control water.
- All eggs were counted, inspected as during the main test, and 20 to 50 fertile eggs were allowed to hatch from each spawning.
- After the sixteenth day, the pH was depressed to 4.5 over a 24 h period and was maintained at this level for 7 d.
- The pH was not adjusted for the remaining 14 d of the experimental period.
Hardness:
28 mg CaCO3
Test temperature:
26 ± 0.5 °C
pH:
- Test systems: pH 4.5, 5.0, 6.0 (nominal)
- Control: pH 6.8 (nominal)
Dissolved oxygen:
Maintained at > 90 % saturation
Salinity:
Not applicable
Nominal and measured concentrations:
See pH conditions (above)
Details on test conditions:
- A mobile toxicity laboratory was established at Lake Panache (Penage) latitude 46 ° 16.4 N, longitude 81 ° 20.6 W was established at Lake Panache (Penage).
- The temperature of incoming lake water pumped from a submerged intake (3 m) was raised to test temperature and maintained at that level in each exposure tank.
- A 15 h light photoperiod with dawn-dusk simulation was used throughout the experimental period.
- The pH of incoming lake water was depressed by addition of 0.1 N sulphuric acid stock injected by an automatic pipettor (Oxford 472A) into the mixing cell of a modified five channel proportional diluter. The diluter discharged 1L of water per channel every 2 min and the flow from each channel was split equally between two exposure tanks. A controller monitor continuously recorded pH in one of the exposure tanks with the lowest nominal pH and provided feedback to the acid injection system. The pH was thus maintained at 4.5 ± 0.1 in those tanks.
- The 50 L stainless steel exposure tanks contained two squat A-shaped stainless steel mesh spawning mats wrapped with green orlon wool and a green transparent Plexiglas partial barrier was placed on one side of each mat to aid the males in establishing spawning territory.
- A spawning event was recorded when eggs were found on a mat.
- Eggs were picked each morning, inspected under a stereo microscope, and infertile or abnormal eggs were removed.
- Fertile eggs were considered those undergoing active embryological development, devoid of abnormalities such as reduced yolk size, lack of blastodisc, or opaque materials in association with the blastodisc or embryo.
- Fertile eggs were collected, placed in floating nylon mesh baskets, and returned to their respective tanks to hatch.
- All eggs (to a maximum of 50) were placed in a hatching basket provided that there were at least 20 eggs in the spawn.
- When spawning was inhibited at lower pH values, excess control eggs were incubated in those affected tanks.
- Spawning was allowed to continue for 20 d after nominal pH levels were reached and, at the end of this period, the adults were sacrificed and their gonads preserved in Bouin's fixitive for later analysis.
- All fry that hatched were counted daily and discarded except for fry hatching 2 to 4 d prior to the sacrifice of the adults. These fry were kept in their hatching baskets and continually fed a diet of newly-hatched brine shrimp (Artemia sp) until the last spawning day.
- Two groups of fry (each containing 25 individuals) were selected randomly from the composite of all hatched fry in each treatment.
- Each group was then returned to one of its respective treatment exposure tanks from which all spawning mats and barriers had been removed.
- This action signified day 1 of the fry growth rate experiment and newly hatched brine shrimp continued to be available to the fry as food.
- After 3, 17, 24, 38 and 45 d, the fry from each tank were removed, placed in a white porcelain bowl with sloping sides, and photographed against a standard measure.


Reference substance (positive control):
no
Duration:
65 d
Dose descriptor:
NOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
nominal
Conc. based on:
other: pH
Basis for effect:
other: fry growth
Remarks on result:
other: Based on LOEC 20% of 0.049 mg/L divided by 2 (equivalent to pH 6)
Details on results:
- pH stability of each treatment group was maintained within 0.13 units at nominal values over the exposure periods.
- Adult mortality occurred principally within the first 20 d of exposure and was about 7 % (one female) in the control group, increasing to 14 % at pH 5.5, 21 % at pH 5.5 and 64 % at pH 4.5.
- All treatment groups commenced spawning within 3 to 4 d of one another and egg production and fertility at the end of both the 10 d acclimation period and the 5 d pH depression period were equivalent in all groups (no correlation to assigned pH).
- Exposure to depressed pH reduced the total number of spawnings in each group compared to controls (P < 0.05) except for pH 6.0 (P > 0.05).
- No spawnings occurred at pH 4.5.
- Spawning frequency overall was observed to decrease directly as the pH was reduced (P < 0.05).
- A significant direct correlation (P < 0.05) was established between total eggs produced and pH.
- Mean daily egg output was significantly impaired (P < 0.05) at each pH level compared to controls.
- Combining data for spawning frequency and total eggs produced shows that mean spawning size (number of eggs produced per spawn) correlated directly with depressed pH (P < 0.05).
- Mean spawning size was significantly reduced compared to controls (P < 0.05) at the pH 5.5 level only.
- The number of fertile eggs was reduced (P < 0.05) at every exposure level compared to controls and the regression related well (P < 0.05) with a decline in pH.
- Egg characteristics used to determine infertility such as reduced yolk size and development of opaque areas were probably true abnormalities because exposure time to depressed pH after egg deposition was always < 24 h.
- hatchability of eggs exposed to depressed pH was variable, ranging from 17 to 56 % and was not indicative of overall trends. However, hatching success was somewhat less during pH 5.0 exposure.
- Eggs collected from control tanks and incubated in pH 5.0 and 4.5 hatched with 82 and 87 % success respectively. This suggests that the parental contribution to egg quality may be an important factor in estimating the extent of environmental effects.
- The control fry that hatched under conditions of depressed pH died within 24 to 56 h.
- The majority of fry mortality in the control and pH 6.0 groups took place within the first week after hatching whilst mortality continued at pH 5.5 and pH 5.0 for more than 3 w.
- Mortalities were not significantly different between control and pH 6.0 treatments but were significantly different (P < 0.05) under pH 5.5 and pH 5.0 conditions.
- Final mean weights of the 45 d fry were significantly less (P < 0.05) after exposure to all pH treatments compared to controls and the decrease was directly related (P < 0.05) to pH depression.
- The correlation coefficient (r = 0.99) of the final mean fry weight and the final mean fry dorsal image area at the different pH values was significant (P < 0.05). A change in the dorsal image area was therefore considered representative of a change in growth.
-Growth rates indicated by changing dorsal image area of the fry held under control and pH 6.0 conditions were similar (P > 0.05) while the growth rates of fry held under pH 5.5 and pH 5.0 conditions were considerably reduced (P < 0.05) compared to controls.
- A significant decrease in growth rate (P < 0.05) between fry in pH 5.5 and pH 5.0 conditions was also evident.
- Breeding communities that were allowed to spawn for 16 d and were then exposed to pH 4.5 for 7 d abruptly stopped spawning for the entire 7 d period. One spawning occurred 7 d after normal lake water conditions resumed and a second was observed on day 9 of the final 14 d period. The mean daily output was reduced from 78 eggs after 22 spawnings before pH depression to 4.7 eggs from 2 spawnings after depression. The total number of eggs produced was 1,249 before pH depression compared to 66 afterwards.

Reported statistics and error estimates:
- Fish measurements were taken from enlarged photographs and dorsal image areas were calculated.
- These data, when subjected to to a logarithmic transformation, were linear with respect to time and were used for growth analysis.
- Spawning frequency, egg fertility, and mortality data were subjected to Chi-square analysis.
- Egg production data were tested for significance by analysis of variance and independent t-distribution.
- Fry growth rate data were compared by analysis of covariance and the correlation of all reproductive responses with declining pH was tested by regression analysis.

JUSTIFICATION FOR USE OF READ-ACROSS DATA

See comparison of overall physico-chemical and toxicity profiles for target and source chemicals in the data matrix (attached).

WATER QUALITY OF LAKE PANACHE

Parameter Measured

Result

pH

6.8

Sulphate

23.1 mg/L

Conductivity (UMHOHS)

81.5

Hardness (as CaCO3)

28 mg/L

Alkalinity (as CaCO3)

11.25 mg/L

Aluminium

< 0.055 mg/L

Cadmium

< 0.001 mg/L

Chromium

< 0.011 mg/L

Copper

0.006 mg/L

Iron

0.016 mg/L

Lead

< 0.006 mg/L

Nickel

0.033 mg/L

Zinc

0.013 mg/L

REPRODUCTIVE RESPONSE OF FLAGFISH EXPOSED TO DEPRESSED pH FOR 20 DAYS

Parameter

Nominal pH

Correlation with pH

6.8 (control)

6.0

5.5*

5.0

4.5

Adult mortalities

1

0

3

2

11

-

Spawnings

32

24

15**

7**

1**

P < 0.05

Total eggs

2,467

1,277

557

319

1

P < 0.05

Mean daily output

61.7

31.9**

13.9**

8.0**

0**

P < 0.05

Mean spawning size

77.1

53.2

37.1**

45.6

1**

P < 0.05

% Fertile eggs

90.1

78.6**

68.0**

55.2**

-

P < 0.05

% Hatching of fertile eggs

44.4

56.5

51.1

17.0

-

P > 0.05

*

Printing error in original paper states value as pH 5.0

**

P < 0.05 compared to control

SURVIVAL, FINAL WEIGHTS AND DORSAL IMAGE AREA OF FLAGFISH EXPOSED TO DEPRESSED pH FOR 45 DAYS

Treatment

Control

pH 6.0

pH 5.5

pH 5.0

Correlation with pH

Total surviving fry (N = 50)

37

43

20

2

P < 0.05

Final mean weight of fry (GMS)

0.55

0.42*

0.26*

0.075*

P < 0.05

Final mean dorsal image area (mm2)

123.7

100.3*

71.5*

37.7*

P < 0.05

*

P < 0.05 compared to controls

Validity criteria fulfilled:
not applicable
Conclusions:
The No Observed Effect Concentration (NOEC) has been calculated as 0.025 mg/L (equivalent to pH 6). The order of sensitivity of flagfish life stages at the 50 % reduction level was egg production > fry survival > fry growth > egg fertility.

Description of key information

NOEC 0.025 mg/L (pre-GLP investigation of reproduction, growth and survival were investigated in Jordanella floridae)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.025 mg/L

Additional information

Sulphuric acid, peroxomonosulphuric acid and hydrogen peroxide are each expected to contribute to the physico-chemical properties of the multi-constituent substance. However, significant toxicological effects are likely to be masked in the multi-constituent substance by its corrosive nature. It is therefore considered appropriate to read-across to the main constituent, sulphuric acid, when considering aquatic toxicity (NOEC equates to pH 6.0).

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