Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (5-7 Na:1)

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key 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 DTPMP-H

Treatment concentration (mg active acid/L)			No. 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).

Reference 2

Endpoint:

fish early-life stage toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to Annex 4 of the CSR and IUCLID Section 13 for justification of read-across within the DTPMP category.

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

Description of key information

(60 d) NOEC 25.6 mg active acid/L (r-a) Salmo gairdneri (new name: Oncorhynchus mykiss).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

25.6 mg/L

Additional information

No data on long term toxicity of DTPMP Na salts to fish are available. However,a long-term fish toxicity test result is available with DTPMP acid (ABC 1980). A 60-day NOEC of 25.6 mg active acid/l based on fry weight, has been determined for early-life stages of O. mykiss,indicating a low order of toxicity. The test satisfied the requirements for achieving a reliability rating of 1. This is the only reliable value and has been selected as key.

The results of tests conducted on DTPMP and its salts are directly comparable, because the substance will fully dissociate in solution and the ionisation state will depend only on the pH of the test medium. The counterion is not considered to contribute to effects in fish. At environmentally-relevant pH values, DTPMP will be ionised typically 6-8 times, and will form stable complexes with metal ions. Here, the value of 25.6 mg equivalent active acid/L may be directly read-across.

The acid and salts in the DTPMP category are freely soluble in water and, therefore, the DTPMP anion is fully dissociated from its cations when in solution. Under any given conditions, the degree of ionisation of the DTPMP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was DTPMP-H, DTPMP (1-3Na), DTPMP (5-7Na), DTPMP (4-8K), DTPMP (xNH4) or another salt of DTPMP.

 

Therefore, when a salt of DTPMP is introduced into test media or the environment, the following is present (separately):

1. DTPMP is present as DTPMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether DTPMP-H, DTPMP (1-3Na), DTPMP (5-7Na), DTPMP (4-8K), DTPMP (xNH4), or another salt was used for testing.

2. Disassociated ammonium, potassium or sodium cations. The amount of ammonium, potassium or sodium present depends on which salt was added.

3. Divalent and trivalent cations have much higher stability constants for binding with DTPMP than the sodium, potassium or ammonium ions so would preferentially replace them. These ions include calcium (Ca²⁺), magnesium (Mg²⁺) and iron (Fe³⁺). Therefore, the presence of these in the environment or in biological fluids or from dietary sources would result in the formation of DTPMP-dication (e.g. DTPMP-Ca, DTPMP-Mg) and DTPMP-trication (e.g. DTPMP-Fe) complexes in solution, irrespective of the starting substance/test material.