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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1979-09-28 to 1979-12-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. Read-across.
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 Unpublished manuscript.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 Dequest 2000 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 frys.


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 uhmos/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 Dequest 2000 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 Dequest 2000 for 60 days was not significantly affected. See Table 1 for details.

- Numbers hatched:: Hatchability of rainbow trout eggs continuously exposed to Dequest 2000 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 Dequest 2000. 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 vailidity criteria for the study was 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.
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1979-09-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to a test protocol that is comparable to a national standard method, in compliance with GLP and analytical monitoring was carried out. Read-across
Qualifier:
according to
Guideline:
other: ABC protocol No. 7810, W. Adams, Monsanto Chemical Industries
Deviations:
yes
Remarks:
water quality parameters were measured on days 4 and 7, rather than 1, 5 and 10
Qualifier:
equivalent or similar to
Guideline:
other: ASTM (1979). Standard practice for conducting toxicity tests on the early life stages of fishes. Draft no. 2, September 1979. ASTM Committee E-35.21.
Deviations:
not applicable
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: One tank from each duplicate at each concentrations.

- Sampling method: The concentrations of Dequest 2060 were determined on days 0, 1, 5, 10, 20, 30, 40, 50, 60 and 66. Alternate duplicate tanks were used from one sample period to the next.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: The stock solutions were prepared on a weight:volume basis by dissolving in deionised water and were delivered to the diluter from a Mariotte bottle enclosed in aluminium foil. Test media were prepared and replaced using a Mount and Brungs proportional diluter

- Controls: dilution water only
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM

- Common name: rainbow trout

- Source: Fish eggs were obtained from Spring Creek Trout Hatchery in Lewistown, Montana. The eggs were held for 24 hours at 10 +/- 1 degree C before testing.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS

- Numbers of parental fish (i.e. of females used to provide required number of eggs): not reported

- Method of collection of fertilised eggs: not reported

- Subsequent handling of eggs: not reported


POST-HATCH FEEDING
- Start date: 1) from latter part of the sac-fry stage until day 30 of growth; 2) after growth day 30 and until the termination of the study

- Type/source of feed: 1) brine nauplii in combination with a standard commercial fish food; 2) Rangen's fish food

- Amount given: 1 and 2) ad libitum

- Frequency of feeding: 1) 3/4 times a day; 2) twice daily
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
66 d
Hardness:
255 ppm as CaCO3
Test temperature:
10 +/- 2 degrees C 
pH:
pH values were also measured during the course of the study and were found to decrease with increasing test concentration - a condition seen in control and test samples and related to the acidic nature of the test material. The pH values ranged between 6.1 in the highest test concentration to 8.0 in the lowest. The lowest pH value was not considered to be low enough to affect the health of the fish.
Dissolved oxygen:
range: 40-70%. The range is said by the authors to be adequate for testing according to the US EPA 1975 Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians. Environmental Protection Agency, Ecological Research Series EPA-660/3-75-009, April 1975, 61 p.
Nominal and measured concentrations:
Nominal concentrations were 16, 31, 63, 125 and 250 mg active acid/L
Mean measured test concentrations were 25.6, 34.2, 67.8, 136.2 and 291.2 mg active acid/L
Details on test conditions:
TEST SYSTEM

- Emybro cups (if used, type/material, size, fill volume): the eggs were incubated in cups suspended in the treatment and control water. Cups were made from 7 cm diameter polyethylene tubing with stainless steel screening (16 mesh) welded at the bottom. To ensure exchange of water, the egg cups were oscillated in the test solution and/or water by means of a rocker arm apparatus driven by a 6 rpm electric motor.

- Test vessel: Each test aquarium was divided by a glass partition to provide space for two growth chambers which measured 25x15x10 cm and had stainless steel screening (16 mesh) attached at one end.

- Material, size, headspace, fill volume: glass aquaria measured 36x30x30 cm with a water depth of 24 cm.

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

- Renewal rate of test solution (frequency/flow rate): The test medium in the test chambers was replaced approximately 5.5 times in 24 hours.

- No. of fertilized eggs/embryos per vessel: 200 eggs per concentration (50 in each cup), 20 fry per growth chamber

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: aerated well water

- Alkalinity: 368 ppm as CaCO3

- Conductivity: 50 uhmos/cm


- Culture medium different from test medium: no

- Intervals of water quality measurement: pH, DO and ammonia were measured initially and on sample days 0, 4, 7, 20, 30, 40, 50 60 and 66 during the study


OTHER TEST CONDITIONS

- Adjustment of pH: none reported

- Photoperiod: 16:8 hour light:dark photoperiod 

- Light intensity: eggs were shielded for UV light exposure


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): egg mortality was recorded daily, when hatching commenced the number of embryos hatching was recorded daily until hatching was complete. Survival of fry was monitored at least weekly by visually inspecting each growth chamber, and behavioural or physical changes were recorded. Growth was determined by the photographic method of McKim and Benoit immediately after transfer of the fry to the respective growth chambers and at 15, 30, 45 and 60 days thereafter.


RANGE-FINDING STUDY: not reported


POST-HATCH DETAILS

- No. of hatched eggs (alevins)/treatment released to the test chamber: 20

- Release of alevins from incubation cups to test chamber on day no.: Since embryo hatching was spread out over 10 days, the modal-hatch date was used to establish day 0 for growth sampling periods.


FERTILIZATION SUCCESS STUDY

- Number of eggs used: 50
Reference substance (positive control):
no
Duration:
60 d
Dose descriptor:
NOEC
Effect conc.:
25.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
weight
Duration:
60 d
Dose descriptor:
LOEC
Effect conc.:
34.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
weight
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: Survival of trout fry exposed to concentrations >136.2 mg active acid/l was significantly decreased relative to control.

- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: Percentage of rainbow trout eggs successful hatching when exposed to 291 mg active acid/L Dequest 2060 was significantly lower than the % hatch of the control eggs. Hatchability of trout eggs exposed to other concentrations were not significantly reduced during the study.

- Observations on body length and weight of young and/or exposed parents at one or more time periods: At concentrations 136.2 mg active acid/L and 291.1 mg active acid/L significantly reduced growth, as measured by standard length, of the rainbow trout fry after 30 days exposure. After 45 days and 60 days of exposure to 67.8 mg active acid/L, growth was signficantly reduced. The effects on the weight of the fry were the same as on length at 60 days of exposure.

- Number of healthy fish at end of test: After 45 days exposure to 291 mg active acid/L only one juvenile rainbow trout had survived and no fish survived 60 days continuous exposure to this concentration.

- Type of and number with morphological abnormalities: No trout fry abnormalities were observed during the study. No difference in egg appearance between Dequest 2060 concentrations and the control were observed.
Reported statistics and error estimates:
Analysis of variance combined with a least significant difference test were used to determine concentrations that had significant effects on survival and growth.

Table 1. Number of hatching eggs to eggs exposed to Dequest 2060

Treatment
concentration (mg active acid/l)

No. of eggs at study initiation

Development
rate

Nominal

Measured

Rep

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 10

Dilution control

1

 50

1

4

17

17

24

19

48

48*

 

 

 

2

 50

 -

-

10

15

20

22

48

45*

 

 

 

3

 50

-

-

 6

16

24

17

47

46*

 

 

4

50

-

1

12

19

28

19

49

47*

 

 

16

25.6

1

 50

2

16

26

16

48

48*

 

 

 

 

2

 50

 -

2

 9

10

25

15

46

46*

 

 

 

 

3

 50

 -

2

 5

9

18

18

46

46*

 

 

4

50

-

-

8

13

22

17

48

48*

 

 

31

34.2

1

 50

-

2

10 

17

27

16

50

47*

 

 

2

 50

 -

-

 6

12

26

13

50

43*

 

 

3

 50

 -

-

 5

10

12

22

43

46

 

 

4

50

-

-

4

13

16

13

49

46

 

 

63

67.8

1

 50

 -

1

 13

17

22

16

48

44*

 

 

2

 50

 -

2

 13

19

25

18

44

46*

 

 

3

 50

 -

1

 10

18

21

21

49

49*

 

 

4

50

-

1

4

10

19

19

47

47*

 

 

125

136.2 

1

 50

-

2

 4

11

15

10

34

40

48*

 

2

 50

 -

-

 9

13

13

13

37

37

47*

 

3

 50

 -

-

 8

13

19

10

45

40

48*

 

4

50

-

-

4

13

14

11

40

39

46*

 

250

291.2

1

 50

 -

3

 7

7

13

10

28

30

29

30*

2

 50

 -

4

 10

18

19

7

32

32

34*

 

3

 50

 -

5

 6

12

18

12

38

38*

38*

 

4

50

-

3

12

15

19

8

31

31

35*

 

*indicates that all eggs that are capable of hatching have hatched

 

Table 2. Mean percentage hatch of eggs, mean survival, standard lengths and wet weight of rainbow trout fry exposed to Dequest 2060.

 

Measured Concentration (mg active acid/L)

Mean hatch %

1-15 days

16-30 days

31-45 days

46-60 days

Mean Total wet weight (g)

A,B **

Mean Total wet weight (g)

C,D **

Survival (%)

Mean length (mm)

Survival (%)

Mean length (mm)

Survival (%)

Mean length (mm)

Survival (%)

Mean length (mm)

0

93

98

21

79

27

74

35

71

43

1.3

1.4

25.6

94

99

21

86

27

85

34

84

41

1.9

1.2

34.2

93

94

20

69

26

73

33*

74

40*

1.1*

9.8*

67.8

93

*96

20*

76

27

75

32*

75

35

0.97*

0.89*

136.2

95

98

18*

70

21*

45

26*

35*

28*

0.36*

0.32*

291.2

69*

88*

16*

28*

18*

 

 

 

 

 

 

 

 

* Denotes values significantly different from the control (p = 0.05) using one-way ANOVA and Fisher’s LSD.

** two sets of test chambers.

 

 

Validity criteria fulfilled:
yes
Conclusions:
A reliable 60 day ELS toxicity test has determined a NOEC value of 25.6 mg active acid/L based on fry weight of the freshwater fish Salmo gairdneri (new name: Oncorhynchus mykiss).

Description of key information

There are no studies are available on SPE1415, however there are two reliable (Klimisch 1) long-term toxicity studies in fish conducted on the analogue substances (ATMP and DTPMP). In addition there is one reliable (Klimisch 2) study on another confidential analogue substance. These data are included in this dossier.

One reliable (Klimisch 1) long-term, early-life stage (ELS) study for ATMP with rainbow trout (Onchorhynchus mykiss, formerly known as Salmo gairdneri) is available (McAllister, 1980).  The GLPcompliant study was conducted over 60 days under freshwater flow-through test conditions. The 60day NOEC (weight of fry and length) was determined to be 23 mg/L, based on mean measured test concentrations as ATMP active acid (50%).

One reliable (Klimisch 1) long-term, early-life stage (ELS) study for DTPMP with rainbow trout (Onchorhynchus mykiss, formerly known as Salmo gairdneri) is available (ABC Laboratories, 1980). The GLPcompliant study was conducted over 66 days under freshwater flow-through test conditions. The 60-day NOEC was determined to be 25.6 mg/L based on mean measured test concentrations as DTPMP active acid (50%).

Additional supporting data on another confidential analogue substance is discussed in Additional Information.

Adequate reliable measured data exists for the long-term toxicity to fish to the aminomethylphosphonate analogue substances of SPE1415 (ATMP and DTPMP).  In a conservative approach, the most sensitive study result from these studies has been identified and used to address the hazard endpoint in question. The most sensitive study result has been identified as a reliable study with ATMP (McAllister, 1980) which reports a NOEC for long-term toxicity in fish of 23 mg/L. Consequently this value will be used for the long term toxicity to fish endpoint for SPE1415.

Key value for chemical safety assessment

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

Additional information