1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

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:

key study

Study period:

19 Dec 1986 to 23 Dec 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Fish were exposed to the test item for 96 hours. Observations for mortalities and symptoms of toxicity were made at 0, 24, 48, 72 and 96 hours.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Cyprinodon variegatus

Test type:

static

Water media type:

saltwater

Limit test:

no

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 112 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

Mean measured concentrations of the test substance demonstrated that a concentration gradient (~ 106% of nominal) was maintained throughout the 96 hour test and defined the exposure concentration range (based on mean measured concentrations) as 14 to 112 mg/L (see table 1). Analysis of quality control samples resulted in measured concentrations in the range of 103 to 116% of the nominal fortified values confirming the appropriate precision and quality control was maintained. The theoretical LOD in this study was <0.0376 µg/L for a 100-mL sample.  Mean measured concentrations were used for the calculation and reporting of results.

Table 1: Analytical results

Nominal concentration (mg/L)	Measured concentration at 0 hours	Measured concentration at 96 hours	Mean measured concentration (mg/L)
Control	<4.9	<5.1	<5.1^a
13	15	12	14^a
22	28	22	25^a
36A	33	36	35^b
36B	38	32
60	56	60	58^a
100	114	109	112^a

^a N =2

^b N = 4; The 36 mg CGA-131036/L treatment level was analysed in duplicate at each sampling interval (0 and 96 hours)

 

The median lethal concentration (LC₅₀) was defined as the concentration resulting in 50% mortality of the fish in the time period specified. Since no deaths occurred among fish exposed to the test substance, the LC₅₀ values were empirically estimated. The NOEC (No Observed Effect Concentration, defined as the highest tested concentration which did not produce an adverse effect when compared to the control) was also determined. No mortality or symptoms of toxicity were observed in the controls. The mortality data and estimated LC₅₀ values are shown in the table 2.

 

Table 2 Effects of the test substance on the survival of Cyprinodon variegatus

Mean Measured Concentration (mg/L)	Cumulative Percent Mortality (%)
	24 hours	48 hours	72 hours	96 hours
Control	0	0	0	0
14	0	0	0	0
25	0	0^a	0	0
35	0	0^b	0	0
58	0	0^a	0	0
112	0	0^c	0	0
LC50 mg/L	-	-	-	>112
NOEC (mg/L)	-	-	112	112

^a Several fish were lethargic

^b Several fish exhibited a partial loss of equilibrium

^c All fish exhibited a partial loss of equilibrium

Validity criteria

Guideline, batch number of test item, expiry date of test item, validity criteria and LOQ were not reported. However this was not regarded as having significant impact on the outcome of the study. 

Validity criteria fulfilled:

yes

Remarks:

See Validity criteria under 'Any other information on results incl. tables'.

Conclusions:

In a 96-h toxicity study on sheepshead minnow without the use of a standard guideline but followed internal protocol, the 96-h LC50 value was determined to be > 112 mg/L (the highest concentration tested), based on mean measured concentrations.

Executive summary:

The acute toxicity of the test item on sheepshead minnow (Cyprinodon variegatus) was investigated in a static study without the use of a standard guideline, but followed internal protocol. The study was conducted under GLP. Fish were exposed to the test item at nominal concentrations of 0 (blank control), 13, 22, 36, 60 and 100 mg/L (mean measured concentrations : < 5.1, 14, 25, 35, 58 and 112 mg/L, respectively) for 96 hours. Ten fish were introduced into 18.9 L glass aquaria containing 15 L of test solution per treatment and control (no replicates). To check the concentrations of the test item, water samples were taken at 0 and 96 hours and analysed by using HPLC. The test conditions were as follows: temperature 21 - 22 °C, pH 7.3 - 7.9, dissolved oxygen 5.5 - 8.2 mg/L (equivalent to 74 - 111 % of air saturation), salinity of 31‰ and a daily photoperiod of 16 hours with a light intensity of 25 foot candles. Observations for mortalities and symptoms of toxicity were made at 0, 24, 48, 72 and 96 hours.

No mortalities were observed in any of the treatments. Effects on swimming behaviour were observed in some or all of the fish exposed to mean measured concentrations of 25 mg/L and higher after 48 hours of exposure. However, there were no symptoms observed at these treatments during the remainder of the test. There were no mortalities or symptoms of toxicity observed in the blank control. Based on mean measured concentrations, the 96-hour LC₅₀ was determined to be > 112 mg/L, the highest concentration tested.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Oct 2012 to 08 Nov 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-1-1

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken in duplicate at 0 hours (initial value) from the centre of the test vessels from test item concentrations of fresh test solution and after 48 and 96 hours from aged test solution. One replicate was analysed and one replicate was stored as backup.

Vehicle:

no

Details on test solutions:

The necessary amount of test item for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask (See Table 1 in ‘Any other information on material and methods incl. tables’). Test medium was added up to the bench mark and the solution was homogenised by shaking and sonication. The stock solution was applied to the respective aquaria and the test solution was stirred for 24 hours before fish were introduced.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Forellenzucht Schneider, D-67734 Katzweiler, Germany
- Length: 53 ± 3 mm (control fish at the end of the test)
- Weight: 1.35 ± 0.22 g (control fish at the end of the test)
ACCLIMATION
Fish for the main test were obtained from the supplier on 18 July 2012 (acclimatisation > 12 days) and held under testing conditions in 300 L containers with continuous renewal of water (5 - 10 % per day) and permanent aeration of water. The fish were held under identical conditions for the entire pre-experimental period. One day before initiation of the test feeding was stopped.
- Feeding: The fish were fed each day with granular rearing food with approx. 2 % of their body-weight. The food was obtained from the fish supplier.
- Mortality: Following a 48-hour settling-in period, mortality was recorded. No mortality above 5 % was observed throughout the acclimatisation period.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

9°dH corresponding to approx. 160 mg/L (as CaCO3)

Test temperature:

16.1 – 16.9 °C

pH:

7.94 – 8.36

Dissolved oxygen:

≥ 94 % of air saturation

Nominal and measured concentrations:

- Nominal concentrations: 0 (blank control) and 100 mg/L
- Measured concentrations day 0: ND and 114 mg/L, respectively.
- Measured concentrations day 2: ND and 109 mg/L, respectively.
- Measured concentrations day 4: ND and 107 mg/L, respectively
See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 L glass aquaria, filled with 15 L
- Aeration: Continuous aeration of the test tanks with a membrane pump using
a Pasteur pipette
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: max. loading of 1 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Test medium: The test medium was water composed of dechlorinated drinking water and deionised water with a conductivity of 390 µS/cm.
- Measurements of water parameters: The pH-value, temperature and O2 concentration were measured in 24 hour intervals from test solutions. Water hardness of the untreated control was determined at the beginning of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily with 30 min transition periods

EFFECT PARAMETERS MEASURED:
Fish were observed at 0, 3, 6, 24, 48, 72 and 96 hours after test start. If present, fish were considered dead if there was no visible movement (e.g. gill movement), and if touching of the caudal peduncle produced no reaction. Records were made on visible abnormalities such as: loss of equilibrium, swimming behaviour, respiratory function, pigmentation and all other observed events, if present.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

An overview of the results is provided in Table 3 - 5 in 'Any other information on results incl. tables'.
After 96 hours of exposure, no motality or abnormalities of fish was found in the control or treated group. Therefore, the LC50 was determined to be > 100 mg/L and the NOEC was 100 mg/L, based on the nominal concentrations.

Sublethal observations / clinical signs:

Table 3. Mortality during the test

Nominal concentration test item [mg/L]	No. of fish tested	Number of dead fish observed after hours
		3	6	24	48	72	96
Control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

 

Nominal concentration test item [mg/L]	No. of fish tested	% of dead fish observed after hours
		3	6	24	48	72	96
Control	7	0	0	0	0	0	0
100	7	0	0	0	0	0	0

Table 4. Observations during the test

 

Test item [mg/L]	Control					100
Time [h]	0	°	*	#	+	0	°	*	#	+
0	7	0	0	0	0	7	0	0	0	0
3	7	0	0	0	0	7	0	0	0	0
6	7	0	0	0	0	7	0	0	0	0
24	7	0	0	0	0	7	0	0	0	0
48	7	0	0	0	0	7	0	0	0	0
72	7	0	0	0	0	7	0	0	0	0
96	7	0	0	0	0	7	0	0	0	0

0 = no clinical signs

° = unusual behaviour (reduced activity and or orientation to bottom or surface of the test vessels, dark pigmentation)

* = difficulties with maintenance of equilibrium

# = fish upside down with loss of equilibrium, showing only movement of gills as a sign of life

+ = no sign of life

 

Table 5. Effect parameters

Time [h]	NOEC (mortality) [mg/L]	LC50 [mg/L]	95 % confidence limit of LC50 [mg/L]
3	100	> 100	-
6	100	> 100	-
24	100	> 100	-
48	100	> 100	-
72	100	> 100	-
96	100	> 100	-

- Not calculable

Validity criteria

According to the OECD 203 (1992), EC-method C.1 (92/69/EEC) and JMAFF Test Guidelines 2-7-1-1 (2000) this study can be regarded as valid, since

• the mortality in the control did not exceed one fish at the end of the test.
• the dissolved oxygen concentration was at least 60 % of the air saturation value throughout the test.

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in ‘Any other information on results incl. tables’

Conclusions:

In a 96-h toxicity study on Rainbow trout following OECD TG 203, OPPTS 850.1075, EC-Method C.1 and JMAFF 2-7-1-1 guidelines, the 96-h LC50 value was determined to be > 100 mg/L (the highest concentration tested), based on nominal concentrations.

Executive summary:

The toxicity of the test item on Rainbow trout (Oncorhynchus mykiss) was investigated in a static study following OECD TG 203, OPPTS 850.1075, EC-Method C.1 and JMAFF 2-7-1-1 test guidelines, and in compliance with GLP principles. Fish were exposed in a limit test to the test substance at concentrations of 0 (blank control) and 100 mg/L (measured concentrations day 0: ND, 114 mg/L, respectively; Measured concentrations day 2: ND, 109 mg/L, respectively; Measured concentrations day 4: ND, 107 mg/L, respectively) for 96 hours. Seven fish were introduced per treatment and control without the use of replicates. To check the concentrations of the test item, water samples were taken at 0, 48 and 96 hours and analysed by using HPLC-MS/MS. The test conditions were as follows: temperature 16.1 - 16.9 °C, pH 7.94 - 8.36 and dissolved oxygen ≥ 94 % of air saturation. Observations of mortality and abnormalities were made at 0, 3, 6, 24, 48, 72 and 96 hours after the addition of the organisms to the test vessels.

After 96 hours of exposure, no mortality or abnormalities of fish were observed in the control and the treatment. Based on the findings, the 96-h LC50 of the test item was determined to be > 100 mg/L (the highest concentration tested), based on nominal concentrations.

Description of key information

Six short-term toxicity studies on fish (5 in freshwater and 1 in marine water) an 1 study on a major metabolite are available and included. More details are provided in the additional information.

- Freshwater: 96-h LC50 > 100 mg/L (OECD TG 203, Wiech 2012)

- Marine water: 96-h LC50 > 112 mg/L (no guideline followed, Surprenant 1987)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 100 mg/L

Additional information

Studies required in Annex VII and some other studies required for higher tonnage are available. All the studies available have been included in the dossier for this endpoint. The lowest effect value of these studies was considered in the aquatic toxicity classification and PBT assessment. Therefore, all studies are summarized below.

There are six studies available for this endpoint, five on freshwater species and one on marine water species. Five studies were performed according to standard guidelines. Only two of the six studies were performed under GLP. All studies were performed under static conditions and determined the same effect value of the substance on the test fish.

Freshwater: Oncorhynchus mykiss was exposed in a limit test to the test substance at concentrations of 0 (blank control) and 100 mg/L (measured concentrations day 0: ND, 114 mg/L, respectively; Measured concentrations day 2: ND, 109 mg/L, respectively; Measured concentrations day 4: ND, 107 mg/L, respectively) for 96 hours. The 96-h LC50 of the test item was determined to be > 100 mg/L (the highest concentration tested), based on nominal concentrations (Wiech 2012). A second study with Oncorhynchus mykiss, one study with Cyprinus carpio, one study with Lepomis macrochirus, and one study with Ictalurus punctatus were exposed to the test item at nominal concentrations of 10, 18, 32, 58 and 100 mg/L. Measured concentrations were in agreement with nominal and remained stable during the exposure period. The 96-h LC50 was determined to be > 100 mg/L for all four species (Rufli 1984a, 1984b, 1984c and 1984d). None of the 4 studies were conducted according to GLP and a high vehicle concentration (553 mg/L DMSO) was used.

Marine water: Cyprinodon variegatus was exposed to the test item at nominal concentrations of 0 (blank control), 13, 22, 36, 60 and 100 mg/L (mean measured concentrations : < 5.1, 14, 25, 35, 58 and 112 mg/L, respectively) for 96 hours. Based on mean measured concentrations, the 96-hour LC50 was determined to be > 112 mg/L, the highest concentration tested (Surprenant 1987).

One study of a major degradation product on Oncorhynchus mykiss is also included. Based on mean measured concentrations, the 96h LC50 was determined to be > 200 mg/L (Rufli 1984e) showing the lower toxicity to fish of this metabolite.