Reaction mass of [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid and Phosphonic acid, P-[(tetrahydro-2-hydroxy-2-oxido-4H-1,4,2-oxazaphosphorin-4-yl)methyl]-

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Annex 5 of the CSR or IUCLID Section 13 for justification of read-across from the ATMP and DTPMP categories to the HEBMP category.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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

Remarks on result:

other: read-across from DTPMP-H

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

Remarks on result:

other: read-across from DTPMP-H

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

Remarks on result:

other: read-across from ATMP-H

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

Remarks on result:

other: read-across from ATMP-H

Reference 2

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

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 Unpublished manuscript.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

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 test material for 60 days was not significantly affected. See Table 1 for details.

- Numbers hatched:: Hatchability of rainbow trout eggs continuously exposed to 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:

A 60-d NOEC of 23 mg/L (as active acid) has been determined for the effects of the test substance on the growth of the freshwater fish Oncorhynchus mykiss.

Reference 3

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 1979-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

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

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

- Embryo 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 insure 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 aquaria 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

- Begin of post-hatch period:

- 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

- Removal of eggs to check the embryonic development on day no.:

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	9	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

No data are available for the registration substance. Data have been read-across from two structurally analogous substances, ATMP-H and DTPMP-H.

NOEC (60 d) 23 mg active acid/L for ATMP-H, based on fry growth (length and weight) of Oncorhynchus mykiss (key).

NOEC (60 d) 25.6 mg active acid/L for DTPMP-H, based on fry weight of Oncorhynchus mykiss (supporting). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

23 mg/L

Additional information

 There are no data available for HEBMP-H or HEBMP-xNa, therefore data have been read-across from two structurally analogous substances, ATMP-H and DTPMP-H.

 

A reliable 60-day NOEC value of 23 mg active acid/L has been determined for the effects of the read-across substance, ATMP-H, on growth (as fry length and weight) of the freshwater fish Oncorhynchus mykiss (ABC, 1980b). The study was conducted according to appropriate test guidelines, in compliance with GLP and assigned a reliability value of 1. As the lower of the two NOEC values, this study was selected as key.

 

A reliable 60-day NOEC value of 25.6 mg active acid/L based on fry weight, has been determined for the effects of the read-across substance, DTPMP-H, on the weight of the early-life stages of the freshwater fish, Onchorhynchus mykiss, indicating a low order of toxicity (ABC, 1980a). The test was conducted according to appropriate test guidelines, in compliance with GLP and assigned a reliability value of 1.

 

 

For information on the read-across from DTPMP-H and ATMP-H, to the HEBMP category, please refer to IUCLID Section 13 and Annex 5 of the CSR.