[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1979-09-28 to 1979-12-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

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 Unpublished manuscript.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 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:

A 60-d NOEC of 23 mg/L (as active acid) has been determined for the effects of the test substance on growth of the freshwater fish 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 3 of the CSR and IUCLID Section 13 for justification of read-across within the ATMP category.

Reason / purpose for cross-reference:

read-across source

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

Reference 3

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The study was assigned a reliability score of 1 in a thorough, peer-reviewed report by the Environment Agency (2007). However, the study has been assigned a reliability value of 2, as there is not enough information on control mortality to compare to validity criteria from modern guidelines.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Test A: O. mykiss eggs (591-737 per vessel) were exposed within one day of fertilisation
Test B: O. mykiss (136-174) were exposed when they reached the eyed egg stage.
In both tests, the test conditions (flow-through apparatus, 15L tanks, etc) were the same but the exposure duration varied (73 days for test A and 49 days for test B)

GLP compliance:

no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Test type:

flow-through

Hardness:

263 +/- 2.3 mg/L as CaCO3

Test temperature:

Mean +/- SD: 14.9 +/- 1.1 degrees C. Overall temperature ranged from 11.5 to 17.0 degrees C.

pH:

7.47-7.57

Dissolved oxygen:

>=95% air saturation value during the pre-hatching stages and 92 and 76% in the larval and post-larval periods, respectively.

Nominal and measured concentrations:

Nominal values were not reported for either test.
Test A Measured: 0.14, 2.55, 4.52, 8.00, 15.50, 25.77 mg NH3-N/L
Test B Measured: 0.19, 2.59, 4.74, 8.15, 15.72, 25.92 mg NH3-N/L

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Not described, however holding tanks contained egg holding trays downstream to maintain a gentle flow of water.
- Test vessel: 15 L tanks fitted with egg holding trays
- Type (delete if not applicable): open - opaque lids were fitted but partially removed before the fry began to feed independently
- Material, size, headspace, fill volume: 15L, fill volume not reported
- Aeration: Yes, further details not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): Test solution was delivered as aqueous solutions to free-piston burettes, which were controlled by concentric-siphon dosers.
- Renewal rate of test solution (frequency/flow rate): Not reported
- No. of fertilized eggs/embryos per vessel: Test A: 591 - 737 and Test B: 136-174
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural, unchlorinated, hard borehole water
- Alkalinity: 243 +/- 3.2 mg/L, other details not reported

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted using small additions of carbon dioxide to the aeration flow as necessary.
- Photoperiod and light intensity not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daily measurements were taken of the following: egg, larval and juvenile mortality, time between fertilisation and hatching, time between hatching to full absorption of the yolk and any other abnormalities.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY - not reported

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: undissociated ammonia as N (NH3-N)

Basis for effect:

mortality

Duration:

73 d

Dose descriptor:

NOEC

Remarks:

as calculated by reviewer by LOEC/2

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: undissociated ammonia as N (NH3-N)

Basis for effect:

mortality

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: unionised ammonia as NH3

Basis for effect:

mortality

Duration:

73 d

Dose descriptor:

NOEC

Remarks:

as calculated by reviewer by LOEC/2

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: unionised ammonia as NH3

Basis for effect:

mortality

Details on results:

Mortality was highest in the egg stage, but yolk-sac fry were still vulnerable.
Fish in test B appeared less susceptible to toxicity.
The data for periods to each developmental stage measured did not differ significantly between treatments

Reported statistics and error estimates:

Not reported

Table 1. Biological observations from test A

Concentrations of ammonia as N (mg/L) Mean (SD)		% Mortality (as individuals entering stage)			Total mortality (%)
Total ammonia	Undissociated ammonia	Egg	Yolk-sac fry	Fry
0.14 (0.17)	-	8.8	1.6	2.6	12.6
2.55 (0.41)	0.022 (0.004)	65.2	12.6	4.9	71.1
4.52 (0.043)	0.039 (0.009)	78.5	33.6	2.7	86.1
8.00 (0.49)	0.069 (0.013)	87.3	44.1	0	92.9
15.50 (0.57)	0.128 (0.012)	72.1	26.8	4.7	80.6
25.77 (1.65)	0.219 (0.040)	97.9	61.5	20.0	99.4

Table 2. Biological observations from test B

Concentrations of ammonia as N (mg/L) Mean (SD)		% Mortality (as individuals entering stage)			Total mortality (%)
Total ammonia	Undissociated ammonia	Egg	Yolk-sac fry	Fry
0.19 (0.20)	-	2.2	4.0	3.6	9.5
2.59 (0.39)	0.023 (0.004)	3.3	9.6	11.6	22.7
4.74 (0.041)	0.041 (0.10)	3.2	5.7	2.5	11.0
8.15 (0.49)	0.072 (0.015)	3.4	3.8	3.5	10.3
15.72 (0.53)	0.130 (0.012)	3.4	11.2	8.3	21.3
25.92 (2.28)	0.226 (0.046)	3.3	14.0	27.5	39.7

Validity criteria fulfilled:

not applicable

Remarks:

No guideline followed. Not enough detail on raw data to directly compare to validity criteria from modern test guidelines.

Conclusions:

A 73-day LOEC value of 0.022 mg NH3-N/L (0.027 mg NH3/L) and a 73-day NOEC value of 0.011 mg NH3-N/L (0.014 mg NH3/L) have been determined for the effects on undissociated ammonia of Oncorhynchus mykiss eggs, larvae and fry.

Description of key information

There are no test data for ATMP-xNH₄, however data have been read-across from ATMP-H.

60-day NOEC 23 mg active acid/L, Oncorhynchus mykiss

Ammonia: 73-day NOEC 0.011 mg NH₃-N/L (equivalent to 0.014 mg NH₃/L), O. mykiss

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

23 mg/L

Fresh water fish

Dose descriptor:

NOEC

Remarks:

toxicity data for ammonia

Effect concentration:

0.011 mg/L

Additional information

ATMP-xNH₄ will dissociate when it is released into aqueous environments to yield ATMP and ammonia. No data is available for ATMP ammonium salts, therefore data have been read-across from ATMP-H.

The key study reported a 60-day NOEC value of 23 mg active acid/L for the effects of ATMP-H on the growth of the fish O. mykiss (ABC, 1980), which has been read-across to ATMP-xNH₄.

Ammonia: a 73-day study reports LOEC and NOEC values of 0.022 and 0.011 mg NH₃-N/L for the effects of unionised ammonia on the egg, yolk-sac and juvenile stages of Oncorhynchus mykiss (Solbe et al., 1989). The NOEC value was derived by dividing the reported LOEC value by 2. The values are equivalent to 0.027 and 0.014 mg NH₃/L. The study was reviewed in the report: Environment Agency Proposed EQS for Water Framework Directive Annex VIII Substances: Ammonia (unionised) 2007 (EA, 2007).

ATMP-xNH₄ will dissociate as it is released into the aquatic environment and it is therefore necessary to assess the hazard properties and derive PNECs for ATMP and ammonia separately.

For hazard classification, ATMP-xNH₄ has been assessed based on the available aquatic data for the whole substance.

The influence of the ammonium salt is discussed in IUCLID Section 6.0 Ecotoxicological Information and Section 7.0 of the CSR.

The acid, sodium, potassium and ammonium salts in the ATMP category are freely soluble in water. The ATMP anion can be considered fully dissociated from its sodium, potassium or ammonium cations when in dilute solution. Under any given conditions, the degree of ionisation of the ATMP 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 ATMP-H, ATMP.4Na, ATMP.7K or another salt of ATMP.

 

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

1. ATMP is present as ATMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether ATMP (3-5K) salt, ATMP (3-5Na) salt, ATMP-H (acid form), or another salt was used for dosing.
2. Disassociated potassium, sodium or ammonium cations. The amount of potassium or sodium present depends on which salt was dosed.
3. It should also be noted that divalent and trivalent cations would preferentially replace the sodium or potassium ions. These would include calcium (Ca²⁺), magnesium (Mg²⁺) and iron (Fe³⁺). These cations are more strongly bound by ATMP than potassium, sodium and ammonium. This could result in ATMP-dication (e.g. ATMP-Ca, ATMP-Mg) and ATMP-trication (e.g. ATMP-Fe) complexes being present in solution.

In this context, for the purpose of this assessment, read-across of data within the ATMP Category is considered to be valid.