Phosphonic acid, monoethyl ester, sodium salt (1:1)

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 - 05 Jun 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Qualifier:

according to guideline

Guideline:

other: Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians (USEPA-660/3-75-009)

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Jugend, Famile und Gesundheit, Wiesbaden

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of the freshly prepared and aged (96 hours old) test solutions was performed for all tested concentrations.

Vehicle:

no

Details on test solutions:

Calculations of the aqueous concentrations of test item were based on 22% purity of the test material. Concentrations are given in mg active ingredient/L. The test item was weighted out to a precision of 0.1 mg. A stock solution was prepared dissolving 91.62 g of the test item in test water and making up to a volume of 450mL. Defined amounts of this stock solution were given into the filled corresponding test tanks and stirred vigorously with ascoop. Defined amounts of test water were removed from the tanks prior adding the test solution to retain a total amount of 45L.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Obtained from the trout hatchery S.A. Charles Murgat, Aquaculture, Les Fontaines, F-38270 Beaufort, France on February 23, 2001 and maintained in the fish maintenance room of the Ökotoxikologie of Aventis CropScience GmbH.
- Age at study initiation (mean and range, SD): 5 months
- Length at study initiation (length definition, mean, range and SD): 6 cm (mean)
- Weight at study initiation (mean and range, SD): 3.76 g (mean)
- Feeding during test: none

MAINTENANCE
The fibre-glass tanks for maintaining fish (from February 27 to June 01, 2001) had a length of 340 cm, a width of 85 cm and a height of 73 cm with a water depth of 60cm. The animals were maintained with a mean water flow of 113.4 L/h (max. 126.0, min. 100.0, standard deviation 7.99), a mean temperature of 13.4 degrees Celsius (max. 16.7, min. 12.9, standard deviation 0.91), a mean pH of 7.3 (max. 7.5, min. 7.1, standard deviation 0.14), a mean dissolved oxygen concentration of 10.0 mg/L (max. 11.4, min. 9.20, standard deviation 0.53), a water hardness of 1.31 (max. 1.45, min. 1.22, standard deviation 0.10) mmol Ca2+ and Mg2+ per liter, and an acid binding capacity of 1.93 (max. 2.39, min. 1.69, standard deviation 0.33), mmol HCI per liter .
The water was constantly aerated to maintain oxygen saturation between 80-100% and consisted of a mixture of 50% filtered tap water and 50% deionized water. Temperature, dissolved oxygen, and pH were measured once a week.
Water analyses (total hardness, acid binding capacity, CI2- and Nitrite- content) were conducted at least once a month. 10 days prior to testing there was no mortality in the stock culture. Fish were neither therapeutically nor prophylactically treated.
The fish were fed on working days (Monday to Friday; five times a week) with standard trout food (KRONEN-FISH, Aminostart, Roth GmbH, D-35274 Kirchhain, FRG) at a daily ration of 1.5% of initial fish wet weight. On regular working days the daily ration was divided into two equal portions and given to the fish in two feeds per day, separated by at least 5 hours. At the start of testing the fish were approximately 5 months old. Representative measurements from a sample of ten test individuals revealed an average weight of 3.76 g, and an average length of 6.0 cm. The fish were a little bit longer than recommended. The longest fish was not twice as long as the shortest. Recommendations regarding body length are 6.0 ± 2.0 cm (EU directive 92/69) and 5.0 ± 1.0 cm (OECD203).

ACCLIMATION
As the holding conditions were very similar to the test conditions, the fish were not acclimated to the exact test conditions in order to avoid any disturbances that may change the behaviour of the fish. One day prior to testing the fish were not fed. The test water was aerated to oxygen saturation and maintained at a temperature of 13 ± 1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

12.8 - 13.8 °C

pH:

7.6 - 8.2

Dissolved oxygen:

7.2 - 10.5 mg O2/L

Nominal and measured concentrations:

control, 10, 18, 32, 56, and 100 mg/L (active ingredient: AE C529354, nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Stainless steel tanks of about 60L with the size of 50 x 19 x 64 cm (approximate values of internal width, height, and length). The depth of test water at the start and at the end of testing was between 14.8 cm and 16.5 cm.
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 0.42g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test water was a well aerated mixture of 50% filtered tap water and 50% deionized water. The two types of water were passed through sand- and activated charcoal filters before being mixed together. The test water was aerated with compressed air to oxygen saturation and temperature regulated to achieve the required test temperature.
- Conductivity: 288 - 396 µS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
- Light intensity: wide spectrum fluorescent lights

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality, behaviour on days 1, 2, 3, and 4

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: equivalent to LC50 >452 mg AEC529354 technical concentrate 22.1%/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: equivalent to NOEC <452 mg AEC529354 technical concentrate 22.1%/L

Details on results:

- Other biological observations: Tetanus, ceased and/or inverted swimming were observed as intoxication symptoms in the treatment levels of 10, 32 and 100mg/L. A low mortality of 10 and 20% occurred in the treatment levels of 10 and 32mg/L, respectively. Therefore, the concentration without mortality and without any intoxication symptoms (NOEC) could not be determined but is below 10mg AE C529354/L.

- Observations on body length and weight: At the end of the study the length of the fish used in the study ranged between 4.9 and 6.9 cm length. Thus, some fish were slightly larger than recommended by the OECD guideline 203 (4.0 - 6.0 cm) but met the recommendations by 92/69/EWG (4.0 - 8.0 cm). The allocation of fish to treatments was performed without conscious bias, and the distribution of fish in excess of 6 cm length was similar across treatments. Thus this deviation from the OECD guideline is not considered significant enough to have affected outcome of this study.

Sublethal observations / clinical signs:

Results of chemical analysis:

Analyses of freshly prepared water for AE C529354 resulted in test item concentrations ranging from 84.6% to 114.5% of nominal values. Analyses of aged water (96h) for AE C529354 at experimental termination resulted in test item concentrations ranging from 79.5% to 111.0% of nominal values. The mean measured values over the time of exposure ranged from 86.8% to 106.7%. At the highest treatment level only the aged water was analysed and revealed a concentration of 111.0% of nominal. Since all analysed concentrations of AE C529354 ranged within ± 20% of nominal at the start and the end of the study, all effect concentrations were based on nominal initial test concentrations.

Table 1: Mortality and intoxication symptoms

Conc. (mg/L)	After 24 hours					After 48 hours					After 72 hours					After 96 hours
	%M	(#/#)			IS	%M	(#/#)			IS	%M	(#/#)			IS	%M	(#/#)			IS
Control	0		(0/10)	-		0		(0/10)	-		0		(0/10)	-		0		(0/10)	-
10	0		(0/10)	-		0		(0/10)	S2/1, S3/1		10		(1/10)	-		10		(1/10)	-
18	0		(0/10)	-		0		(0/10)	-		0		(0/10)	-		0		(0/10)	-
32	0		(0/10)	-		0		(0/10)	S1/2		10		(1/10)	S1/3		20		(2/10)	S1/3
56	0		(0/10)	-		0		(0/10)	-		0		(0/10)	-		0		(0/10)	-
100	0		(0/10)	-		0		(0/10)	S1/1		0		(0/10)	S1/1		0		(0/10)	S1/1

(#/#) = number dead / number treated

%M = percent mortality

IS = intoxication symptom

S1/# = tetanus / number of fish with symptom

S2/# = inverted swimming / number of fish with symptom

S3/# = ceased swimming / number of fish with symptom

Table 2: Validity criteria OECD 203

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0% of the control fish died at the end of the test	Yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	The dissolved oxygen concentration was at least 69% of the air saturation value throughout the test (based on 10.5 mg O2/L maximum saturation at 13 °C)	Yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	The recovery of the test item ranged from 79.5% to 111%.	Yes

Validity criteria fulfilled:

yes

Remarks:

see Table 2 in 'any other information on results incl. tables'

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 - 23 Aug 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: negative control and 100 mg/L
- Sampling method: Samples were taken at least in duplicate at 0, 24, 48, 72, and 96 hours
- Sample storage conditions before analysis: One sample was analysed after pre-treatment, the remaining samples were retained in case further analysis would be required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test solution was prepared by directly adding appropriate amounts of Fosetyl-Na in dilution water and stirring for 30 min. For the limit concentration of 100 mg/L 0.9224 g of Fosetyl-Na was added to 9 L of dilution water.
- Controls: dilution water as negative control

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: rare minnow
- Strain: batch No.: F20140715
- Source: obtained as fry from a fish supplier of Institute of Hydrobiology, Chinese Academy of Sciences
- Length at study initiation (length definition, mean, range and SD): mean = 2.57 cm (RSD = 7.22%)
- Weight at study initiation (mean and range, SD): mean = 0.252 g wet weight (RSD = 6.91%)

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: the fish were fed on proprietary fish food (>36% crude protein, >2% crude fat, < 3% crude fibre, < 12% crude ash, < 10% moisture)
- Feeding frequency during acclimation: daily
- Health during acclimation (any mortality observed): No mortality in 7 days following after 48-h settling-in period


FEEDING DURING TEST (as applicable) : no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

115 - 123 mg CaCO3/L

Test temperature:

23.0 - 23.1 °C

pH:

7.45 - 7.79

Dissolved oxygen:

68 - 89% of the air saturation value

Nominal and measured concentrations:

- Nominal concentrations: control, 100 mg/L
- Measured concentrations control (0, 24, 48, 72, 96 h): < LOD, < LOD, < LOD, < LOD, < LOD mg/L; mean = < LOD
- Measured concentrations 100 mg/L (0, 24, 48, 72, 96 h): 106, 102, 99.8, 101, 100 mg/L; mean = 102 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass tanks with a sealable inert lid
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 5 L
- Aeration: no
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Biomass loading rate: 0.5 - 0.7 g fish (wet weight)/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: good quality tap water hich has been dechlorinated for at least 12 hours
- Total organic carbon: 1.91 mg/L
- Metals: < 0.001 mg/L (As, Cd, Pb, Co, Ag); < 0.0002 mg/L (Hg); 0.001 mg/L Ni; 0.002 mg/L Cu; < 0.004 mg/L Cr6+; < 0.03 mg/L Fe; 0.25 mg/L Zn
- Pesticides: < 0.001mg/L (malathion, methyl parathion, and parathion)
- Alkalinity: pH = 7.55
- Culture medium different from test medium: no
- Intervals of water quality measurement: the characteristics of the dilution water are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours darkness
- Light intensity: 1000 - 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and abnormal behaviour like inactivity and strange swimming pattern were recorded at 24, 48, 72, and 96 hours. Fish were considered dead if there was no visible movement (e.g. gill movement) and if touching of the caudal peduncle produces no reaction. Temperature, pH, and dissolved oxygen were measured and recorded daily.

TEST CONCENTRATIONS
- Range finding study : yes
- Test concentrations: control, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: no dead fish at any tested concentration after 96 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is used as reference substance at least once for every batch of fish

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Abnormal responses: no abnormal behaviour was observed throughout the entire test duration

Results with reference substance (positive control):

- Results with reference substance valid? yes
- LC50: the 24-h LC50 with potassium dichromate was 269 mg/L (95% CL: 220 - 330 mg/L)

Reported statistics and error estimates:

Trimmed Spearman-Karber Method (Version 1.5, US EPA)were used to calculate the LC50 and the 95% confidence limits. Due to its low toxicity to fish in this study, only LC50 and 95% confidence limits of reference substance was calculated by this method.

Sublethal observations / clinical signs:

Table 1: Mortality of Gobiocypris rarus after a 96h exposure to Fosetyl-Na

Nominal Concentration [mg/l]	Initial number of fish	Mortality
		24h	48h	72h	96h	∑
Control	7	0	0	0	0	0
	7	0	0	0	0	0
	7	0	0	0	0	0
100	7	0	0	0	0	0
	7	0	0	0	0	0
	7	0	0	0	0	0

 

Table 2: Validity criteria for OECD 203.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0%	Yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	68 – 89% air saturation throughout the test	Yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	The measured concentrations were within ± 20% of nominal concentrations.	Yes

 

Validity criteria fulfilled:

yes

Remarks:

see Table 2 in 'any other information on results incl. tables'

Description of key information

LC50 (96 h) > 100 mg a.i. (Fosetyl-Sodium)/L for Onchorhynchus mykiss (OECD 203) (equivalent to > 452 mg Fosetyl-Sodium technical concentrate 22.1%/L)

LC50 (96 h) > 100 mg/L for Gobiocypris rarus (OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L

Additional information

A GLP study according to OECD guideline 203 on the acute toxicity to the rainbow trout (Oncorhynchus mykiss) was performed with Fosetyl-sodium (AE C529354; technical concentrate; Code: AE C529354 00 TK22 A101; containing 22.1% w/w Fosetyl-Sodium) under static conditions.

Five months old trouts were exposed to the nominal concentrations of 10, 18, 32, 56, and 100 mg Fosetyl-Sodium/L and an untreated control at 13 ± 1 °C for 96 hours. The low purity (22.1%) of the technical concentrate was taken into consideration. The test item was dissolved in test water. No additional solvent was used. The test water was not aerated during the time of testing.

Chemical analysis of the freshly prepared and aged (96 hours old) test solutions was performed for all tested concentrations. Samples were taken from the test chambers and analysed for the test item using High Performance Liquid Chromatography with ultraviolet detection (HPLC-UV). The concentrations were diluted prior analyses.

Analyses of freshly prepared water for the test item resulted in concentrations ranging from 84.6% to 114.5% of nominal values. Analyses of aged water (96 h) for the test item at experimental termination resulted in concentrations ranging from 79.5% to 111.0% of nominal values. The mean measured values over the time of exposure ranged from 86.8% to 106.7%. At the highest treatment level only the aged water was analysed and revealed a concentration of 111.0% of nominal. Since all analysed concentrations of the test substance ranged within ± 20% of nominal at the start and the end of the study, all effect concentrations were based on nominal initial test concentrations. Tetanus, ceased and/or inverted swimming were observed as intoxication symptoms in the treatment levels of 10, 32 and 100 mg/L. Therefore, the concentration without mortality and without any intoxication symptoms (NOEC) could not be determined but is below 10 mg Fosetyl-Sodium/L.

A low mortality of 10 and 20% occurred in the treatment levels of 10 and 32 mg/L, respectively. The concentration of test substance lethal to 50% of the test animals (LC50) after 24, 48, 72 and 96 hours test duration was >100 mg Fosetyl-Sodium/L (equivalent to > 452 mg technical concentrate 22.1% / L).

Data of a second GLP study is available which was conducted according to OECD guideline 203 on the acute toxicity of Fosetyl-Sodium (analytical purity: 97.6%) to the rare minnow (Gobiocypris rarus) under static conditions. Fish were exposed to a limit concentration of 100 mg/L and a negative control. The test item was directly dissolved in test water and no additional solvent was used. The test water was not aerated during the time of testing. Chemical analysis of the freshly prepared and aged (0, 24, 48, 72, and 96 hours old) test solutions was perfomrmed by using HPLC-MS/MS.

The results revealed that measured concentrations of old and aged solutions were within +/- 20% of nominal concentrations. Therefore, results were based on nominal concentrations. No mortality or abnormal behaviour was observed in control and treated fish at any time. Thus, the 96-hour LC50 was > 100 mg/L nominal concentration. The dissolved oxygen concentration ranged from 68 – 89% of the air saturation value throughout the test. Therefore, the test was considered valid according to the validity criteria given in OECD guideline 203.