Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 14/12/04; Experimental Completion Date: 18/12/04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

other: The test procedure is based on test guideline PARCOM 1995 Part B Protocol for a Fish Acute Toxicity Test (modified OECD 203 Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Samples were not taken for analytical measurement of test substance concentration in the test samples.

Vehicle:

no

Details on test solutions:

The test substance as thoroughly homogenised before use. The test substance was soluble in water, and the sample preparation method for water soluble test substances was followed. The water accommodated fraction (WAF) of the test substance was tested. Standard sample preparation methods are described below:

Water soluble test substances:
- Add the test substance directly to a beaker containing 0.5 litre of seawater. Add 3 times the final test substance concentration.
- Mix the solutions on magnetic stirrers for >30 minutes. Cover the top of the vessels with aluminium foil or parafilm to minimise volatilisation.
- Add the contents of the beaker to 2 litre of clean seawater and rinse the beaker with 0.5 l of seawater. The total volume of seawater is now 3 l.
- Prepare the required test concentrations using graduated pipettes and volumetric measuring flasks, or scale.

The following test concentrations (nominal concentrations of whole test substance) were used for the definitive test: 100, 300 and 1000 mg/l.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Source: Sea Platation Engineering Inc.
- Age at study initiation (mean and range, SD): Juvenile
- Length at study initiation (length definition, mean, range and SD): 1.3 cm - 2.2 cm; average 1.9 cm
- Weight at study initiation (mean and range, SD): 0.0512 g - 0.1928 g; average 0.1340 g


ACCLIMATION
Holding conditions: Synthetic seawater; 17.6 - 22.4ºC, salinity: 33%, dissolved oxygen >60%; flow through
On arrival at the laboratory, the fish were transferred to a temperature room to allow temperature acclimatisation. Temperature in the fish containers and the acclimatisation tank were measured. The fish were held in a seawater flow-through system, and pH, salinity, O2 saturation and temperature were measured within one hour of arrival and subsequently on every working day. Aeration was supplied and the fish were fed at least once per working day. Mortality during the first 7 days after arrival (after the two day settling-in period) was 0%. This was within the valid range.

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable.

Hardness:

Not stated.

Test temperature:

Temperature in test solutions ranged from 19.4 - 20.2ºC

pH:

pH in test solutions ranged from 6.0 - 8.1

Dissolved oxygen:

Dissolved oxygen in test solutions was ≥90%

Salinity:

Salinity in test solutions ranged from 3.2 - 3.3%

Nominal and measured concentrations:

definitive test: 100, 300 and 1000 mg/l (nominal concentrations of whole test substance)

Details on test conditions:

TEST SYSTEM
- Renewal rate of test solution (frequency/flow rate): Test media changed after 48 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
Reference seawater used for testing was bought from Bjolsen Akvarium AS and was stored for no longer than 4 weeks at temperatures between 18 and 22ºC

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The fish were inspected at least once per day during the whole test period. Mortalities were recorded at 24, 48, 72 and 96 hours. Fish that were considered dead were removed from the test vessels and mortality was recorded. Behaviour was also observed during the test period.

Measurements of pH, dissolved oxygen, salinity and temperature were carried out daily in all test vessels including the control.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(whole test substance)

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(whole test substance)

Basis for effect:

mortality (fish)

Details on results:

Log test substance concentrations, mortality, LC50 values and variables are shown in Tables 1 and 2.

The LC50 value for the test substance R-7398E salt was found to be >1000 mg/l after 96 hours.

The 95% confidence range could not be calculated.

The NOEC was estimated to be 1000 mg/l.

LC50 values after 24, 48 and 72 hours were all >1000 mg/l.

Results with reference substance (positive control):

A reference test using a single concentration of 2.3 mg/l of 3,5-dichlorophenol was performed. The recorded mortality was 60% after 96 hours.

Sublethal observations / clinical signs:

Table 1. Log Test Substance Concentrations and Mortality after 24, 48, 72 and 96 Hours

Test Conc (mg/l)	Log Test Conc	No. fish per vessel	Mortality 24 h	Mortality 48 h	Mortality 72 h	Mortality 96 h	Cumulative mortality %
Control	-	10	0	0	0	0	0
100	1.0	10	0	0	0	0	0
300	1.48	10	0	0	0	0	0
1000	2.00	10	0	0	0	0	0

Table 2. LC50 values and thr 95% Confidence Limits after 24, 48, 72 and 96 hours for the Test Substance R-7398E salt

Duration (hours)	LC50 (mg/l)	Lower 95% CL (mg/l)	Upper 95% CL (mg/l)
24	>1000	-	-
48	>1000	-	-
72	>1000	-	-
96	>1000	-	-

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 value for the test substance R-7398E salt was found to be >1000 mg/l (nominal whole test substance). The NOEC value was estimated to be 1000 mg/l nominal whole test substance).

Executive summary:

Acute toxicity (96 h LC₅₀: semi-static test) of the test substance R-7398E salt to the juvenile Sheepshead minnow, Cyprinodon variegatus, has been assessed in accordance with test guideline PARCOM 1995 Part B (modified OECD Guideline 203 "Fish, acute toxicity test").

The tested concentrations were 100, 300 and 1000 mg/l (nominal whole test substance).

The 96 hour LC₅₀ value for the test substance R-7398E salt was found to be >1000 mg/l (nominal whole test substance). The 95% confidence limit could not be calculated. The NOEC value was estimated to be 1000 mg/l (nominal whole test substance).

In the test the control parameters for temperature, dissolved oxygen and salinity were within the recommended ranges.

Description of key information

LC₅₀ (96 h) >300 -600 mg active acid/L (nominal) for sodium salt of HEBMP, based on (absence of) mortality of Cyprinodon variegatus.

Key value for chemical safety assessment

Marine water fish

Marine water fish

Dose descriptor:

LC50

Effect concentration:

> 300 - ca. 600 mg/L

Additional information

The study was conducted in a recommended species, in accordance with an appropriate guideline and GLP. Validity criteria were fulfilled. The result is equivalent to >300 mg/l - >600 mg/L active acid equivalent (based on reported active ingredient content).

Whilst the proportion of cyclic and linear constituents was not measured for the tested sample, it is conservatively interpreted that the cyclic constituent could have been present at ca. 50% w/w.