cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Oct 2005 to 23 Mar 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken from the test concentrations and control(s) at test start, weekly thereafter and at test termination. At test start and termination samples were taken from every test vessel. Weekly samples were taken from each replicate per treatment in turn. On each sampling occasion, duplicate samples of approximately 10 mL were taken from the centre of each test vessel. One sample of each replicate was analysed and the second sample was stored as a back up. At application solution renewal time approximately 10 mL of the freshly prepared and old application solutions were taken.

Vehicle:

no

Details on test solutions:

The test item was dissolved in dilution water to yield an application solution of 25 mg/L. Initially the test substance was blended by means of a hand held blender into a small volume of dilution water (approximately 200 mL). This was then made up to 50 L and stirred for approximately 24 hours before use. All application solutions were clear and colourless following this stirring period. The test concentrations were prepared by the dilution of the application solution using a proportional diluter system. Dosing was started prepared approximately 24 hours before the test start in order to allow the test solutions in the vessels to have reached equilibrium. The application solution was renewed weekly. Application solution consumption was monitored by measuring the depth of application solution consumed daily.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Fertilised eggs: The eggs were fertilised at 10:45 am on 20 December, 2005 and transported immediately to the testing facility.
- Feeding during test: Larvae and juveniles were fed once the swim-up phase, defined as active swimming in the water column, commenced (day 43). Larvae and juveniles were fed a commercially available trout starter food, at least twice per day on week days and at least once a day on weekends and public holidays. Surplus food and waste was removed from the test vessels, as required. Feeding was withheld 24 hours prior to measurements at study termination.
- Feed quality: A representative sample of the fish food was analysed for the presence of pesticides, PCBs and toxic metals. None of these compounds were detected at levels considered toxic.

ACCLIMATION
- Acclimation conditions: Upon arrival at the test facility, approximately 13:15 pm, the water temperature of the dilution water containing the eggs was 8.3 °C and by the end of counting the temperature had risen to 13.2°C (14:15 pm). Therefore, the temperature of the water bath was adjusted to allow for approximately 11.5 °C in the vessels (within ± 2°C of the eggs). The following day the temperature of the bath was adjusted back to allow a temperature of 10 ± 2°C in the test vessels.
- Handling: Eggs and newly hatched larvae were handled with wide bore glass dropping tubes. At test termination a fine-mesh dip net was used to transfer fish.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

93 d

Remarks on exposure duration:

Includes 59 days post-hatch

Hardness:

197 - 227 mg/L as CaCO3

Test temperature:

- Day 0: 11.2 to 11.7 °C
- Day 1 - Day 33 (hatching period): 9.7 - 10.5°C
- Day 34 - Day 95 (post hatching): 11.6 - 12.1 °C

pH:

7.54 - 7.99

Dissolved oxygen:

76 - 98% saturation

Conductivity:

390 - 511 μS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 9, 19, 38, 75, 150 and 300 μg/L
- Measured concentration: 0 (control), 5.59, 18.8, 39.1, 72.2, 160 and 305 μg/L, respectively See Table 1 in 'Any other information on materials and methods incl. tables',

Details on test conditions:

TEST SYSTEM
- Test vessel: Approximately 15 L glass aquaria
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- Aeration: From day 73 until test termination, test vessels were aerated via glass diffuser stones placed in each vessel
- Type of flow-through: A modified proportional diluter with an appropriate toxicant metering system was used for the intermittent introduction of test solution (test item and dilution water) into the test vessels. The diluter delivered 12 mL of the application solution per cycle (cycle time 3 minutes) to a mixing chamber. Dilution water was added to give a total volume of 984 mL at a nominal concentration of 0.3 mg/L. This was further diluted to generate the other test concentrations. The diluter was started one day before the test start to allow performance system checks and equilibration. A complete check of the diluter system, consisting of visual observation of a complete water cycle was made once daily. Application solution consumption, water and test solution volumes which were delivered through the diluter, and number of cycles completed were also recorded daily. The flow splitting accuracy to each replicate was measured twice during the study.

- Flow rate: Every diluter cycle delivers 0.5 L of water or test solution to one replicate. The diluter was set to run a cycle every three minutes, therefore, approximately 480 cycles were completed per 24 hours per treatment. As the diluter sequentially feeds the three replicates, over three consecutive cycles each replicate therefore receives the volume of 160 cycles, which corresponds to 80 L of water or test solution per 24 hours. The test vessel working volume was 15 L giving approximately five volume exchange per day.

- Biomass loading rate: 0.32 g/L per 24 hours or instantaneously 1.62 g/L for the control fish (calculated across all replicates at test termination).
- Egg cup incubation: From arrival (day 0) until day 34, eggs were incubated in egg cups. When the assignment of eggs was complete the egg cups were placed in the appropriate test vessel which signified the start of the test. During this 34 days, the eggs were gently moved by vertical oscillation of the egg cups with a rocker arm apparatus.
- Test cups: Glass cylinders (diameter approximately 7 cm) closed at one end by fine stainless steel mesh net.

- No. of eggs per cup: 30. On day 34 (23 January 2006), hatching was estimated to be approximately 90% for the dilution water control. Therefore, larvae from all replicates were counted and transferred into the test vessels. Day 34 was defined as "Hatching day", the day at which the highest number of larvae which hatched in the control replicates on any one day. The majority of eggs in all treatment replicates hatched on the same day. The exposure period lasted for 59 days post-hatch terminating on 23 March 2006.

TEST MEDIUM
- Source/preparation of dilution water: The dilution water was prepared by mixing mains water with deionised water to give a total hardness of 100 - 250 mg CaCO3/L. The mixed water then passes through an activated carbon filter and a UV steriliser to produce the dilution water. Analysis of representative samples of dilution water is conducted on at least a biannual basis for potential toxicants, pesticides, particulate matter, total organic carbon and metals The most recent analysis indicated that the dilution water was of a suitable quality in agreement with the testing guidelines followed

WATER PARAMETERS:
- Temperature, dissolved oxygen and pH: Temperature, dissolved oxygen and pH measurements were recorded at test start and once per week during the experimental phase of the test in all replicates of each treatment. One control vessel (replicate A) was continuously monitored for temperature. On day 0, the temperature of the water bath was adjusted to approximately 11.5 °C to avoid thermal shock of the eggs and then decreased to approximately 10 °C the following day.
- Hardness and conductivity: Hardness and conductivity were measured in all test vessels at test start and termination.

OTHER TEST CONDITIONS
- Lighting regime (before day 41): Embryos were kept in the dark, except during observations, until one week after hatching (day 41).
- Photoperiod (after day 41): 16-hour light: 8-hour dark, with a 30 minute dawn/dusk transition period was maintained throughout the remainder of the test.

EFFECT PARAMETERS MEASURED
- Egg viability assessment: After 14 days the fertilisation control eggs were transferred to a 10% (v/v in dilution water) acetic acid solution. After some minutes the eggs became clear and it was possible to count the number of eggs showing a white line (neural keel). The presence of the neural keel indicated that the egg had been fertilised. The egg viability for the batch was calculated as the number of embryos with neural keel out of the total number of eggs (90 eggs), and expressed as a percentage.
- Hatching: Hatching was estimated visually on a daily basis. During the prehatching phase, egg mortality, defined as opalescent white eggs was recorded daily. Dead eggs were removed when observed.
- Survival: Daily observations (visual inspection) of egg and larval mortality, behaviour and appearance were made and any abnormal effects recorded.
- Others: At test termination, all surviving fish in each replicate were terminally anaesthetised and measured for total length, blotted on paper towels to remove excess moisture, and weighed. Following these measurements the fish were humanely terminated.

Reference substance (positive control):

no

Key result

Duration:

93 d

Dose descriptor:

NOEC

Effect conc.:

0.305 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: hatching success, survival or larval growth

Details on results:

An overview of the results is provided in Table 2 – Table 4 in ‘Any other information on results incl. tables.

- Egg viability: The egg viability of the fertilisation control was 88% (determined after 14 days). This demonstrated the good quality of eggs used.

- Survival at hatching: Egg mortality was low across all treatments. Survival at hatching in the three dilution water control replicates ranged from 83 - 93% and from 70 - 93% in the test substance treatments. There was no statistically significant (two-tailed Fisher's exact test) difference between dilution water control and any test substance treatment for survival at hatching.

- Survival at test end: Larval survival in the three dilution water control replicates ranged from 96 - 100% and from 81 - 100% in the test substance treatments. There was no statistically significant (two tailed Fisher's exact test) difference between dilution water control and any test substance treatment for survival between hatching and test termination.

- Sub-lethal effects (hatching): Hatching started synchronously in all replicates of the dilution water control and the test substance treatments on day 34 ("Hatching day") and was complete by day 35. One egg of the dilution water control failed to hatch but was confirmed dead on day 38. Overall hatching success was good ranging between 80 - 93% and 70 – 93%, for the dilution water control and the test substance treatments, respectively.

- Sub-lethal effects (Deformity): Shortly after hatch, on day 35, several larvae were observed to have kyphotic spinal curvatures (convex bowing of the spine). By day 38 some of these larvae had recovered and exhibited straight spines. However, in the 39.1 µg/L (two replicates), 72.2 µg/L (one replicate) and 160 µg/L (one replicate) deformities persisted. By day 52 it was clear that these individuals were unable to feed. Consequently, deformed larvae were terminated according to UK Home Office regulations. These deaths were treated as mortalities for subsequent data analysis. The spinal deformities are not considered test item related for the following reasons:
• The deformities were not dose related in occurrence or severity
• Overall low incidence
• Likelihood that the deformity is the result of physical damage during hatching (and the noted recovery of some individuals)
On day 87 it was observed that one fish in the highest treatment level (305 µg/L) exhibited open opercula and reddened gill surfaces. This persisted until the termination of the study. Occasionally larvae were observed to be small in comparison to control animals. This effect will not be discussed further as it fully considered in the assessment of growth (length and weight).

- Time to swim up: Swim-up began on day 47 and was complete by day 56 in all treatments.

- Behaviour: No unusual behaviours were noted.

- Length: No statistically significant (two-tailed Dunnett's test) difference in length between control and treatment was found. Therefore, the NOEC for fish length is 305 µg/L (the highest concentration tested).

- Weight: No statistically significant (two-tailed Dunnett' s test) difference in weight between contro1 and treatment was found. Therefore, the NOEC for fish weight is 305 µg/L (the highest concentration tested).

Table 2 Summary of egg and larvae mortality

Mean measured concentration (µg/L)	Replicate	Total number dead eggs	Total number dead larvae	Number of larvae counted at 93-days
Dilution water control	A	6	0	24
	B	2	1	27
	C	6	1	23
5.59	A	7	0	23
	B	6	2	22
	C	9	4	17
18.8	A	3	1	25a
	B	6	1	23
	C	4	0	25a
39.1	A	4	2	24
	B	5	3	22
	C	2	2	26
72.2	A	6	1	23
	D	5	1	24
	C	6	2	22
160	A	3	0	26a
	B	11	2	17
	C	9	1	20
305	A	4	2	23a
	B	4	3	25b
	C	6	0	24

Note: there were no statistically significant differences amongst the dilution water control and any test substance treatment for egg survival (0-34 days) or larval survival (34-93 days) (two-tailed Fisher's exact test).

a. One less larva recovered at the end of the test than expected. One of these discrepancies is explained by an escapee found in the water bath (day 76). lt is likely the others perished and decomposed either in their test vessel or after escaping unnoticed to the water bath. For all data analysis the number recovered on day 93 was used.

b. Two more larvae recovered at the end of the test than expected. Possibly attributable to an error in tile number of eggs plated in the vessel at test start. As the difference in overall mortality is minor the data analysis was conducted assuming 30 eggs at the test start.

Table 3. Hatching success, percent egg survival and percent larval survival

Mean measured concentration (µg/L)	Replicate	Hatching success(%)	Survival day 0 – 34 (%)	Survival day 34 – 93 (%)
Dilution water control	A	80	83	100
	n	93	93	96
	C	80	83	96
	mean	84	86	97
5.59	A	77	77	100
	B	80	80	92
	C	70	70	81
	mean	76	76	91
1 8.8	A	90	90	93
	B	80	80	96
	C	87	87	96
	mean	86	86	95
39.1	A	87	87	92
	B	83	83	88
	C	93	93	93
	mean	88	88	91
72.2	A	80	80	96
	B	83	83	96
	C	80	80	92
	mean	81	81	95
160	A	87	87	100
	B	63	60*	94*
	C	70	70	95
	mean	73	72	96
305	A	87	87	88
	B	87	87	96
	C	80	80	100
	mean	85	85	95

*One larva died on day 34 and so is included in both days 0-34 and 34-93 survival calculations

  Table 4. Summary of fish growth (length and weight) at 93 days

Mean measured concentration (µg/L)	Replicate	Mean total length (mm)	Mean wet weight (mg)
Dilution water control	A	49	1019
	B	48	931
	C	49	1006
5.59	A	48	966
	B	48	929
	C	49	985
18 .8	A	48	919
	B	50	997
	C	49	975
39.1	A	50	982
	B	47	870
	C	47	913
72.2	A	48	929
	B	50	1049
	C	51	1067
160	A	49	945
	B	50	1050
	C	48	895
305	A	46	740
	B	48	814
	C	50	940

Note: there were no statistically significant differences amongst the dilution water control and any test substance treatment for fish weight or length (two-tailed Dunnett's test).

 

Validity criteria fulfilled:

yes

Remarks:

See Validity criteria in 'Any other information on materials and methods incl. tables'

Conclusions:

There were no significant effects of the test substance on hatching success, survival or larval growth. Therefore, the overall study NOEC was 305 μg/L, based on mean measured concentrations.

Executive summary:

The chronic toxicity of the test substance to early life-stage of fish was evaluated in a 93 days (56 days post hatch) flow-through test with rainbow trout (Oncorhynchus mykiss) embryos and larvae. The study followed OECD TG 210 and EPA OPPTS 850.1400 guideline, and in compliance with GLP criteria. The mean measured concentration of the test substance were 5.59,18.8, 39.1, 72.2, 160 and 305 µg/L (measured by a LC-MS/MS; nominal concentrations were 9, 19, 38, 75, 150 and 300 µg/L, respectively). In addition, a dilution water control was also included in the test. The study started from 11.2 to 11.7 °C. During hatching period, the temperature was adjusted to 9.7 - 10.5°C. After hatching (day 34), the temperature was changed to 11.6 - 12.1 °C until the end of the test. In all testing groups, the following conditions were maintained: pH 7.54 - 7.99, dissolved oxygen concentration of 76 - 98% saturation, water hardness of 197 - 227 mg/L (as CaCO3) condition and conductivity 390 - 511μS/cm. The biomass loading rate was 0.32 g/L per 24 hours. Observations were made on survival of organisms at hatch and survival and growth (wet weight and total length) of larvae.

 

The results showed that the egg viability of the fertilisation control was 88% (determined after 14 days). This demonstrated the good quality of eggs used. Egg mortality was low across all treatments. Overall hatching success was good ranging between 80 - 93% and 70 – 93%, for the control and the test substance treatments, respectively. There was no statistically significant difference between control and any test substance treatment for survival at hatching or for survival between hatching and test termination. Shortly after hatch, on day 35, several larvae were observed to have kyphotic spinal curvatures (convex bowing of the spine). By day 38 some of these larvae had recovered and exhibited straight spines. However, in the 39.1 µg/L replicates A and C, 72.2 µg/L replicate A and 160 µg/L replicate B deformities persisted. By day 52 it was clear that these individuals were unable to feed. Consequently, deformed larvae were terminated according to UK Home Office regulations. These deaths were treated as mortalities for subsequent data analysis. On day 87 it was observed that one fish in the highest treatment level (305 µg/L; replicate A) exhibited open opercula and reddened gill surfaces. This persisted until the termination of the study. Occasionally larvae were observed to be small in comparison to control animals. This effect will not be discussed further as it fully considered in the assessment of growth (length and weight). Swim-up began on day 47 and was complete by day 56 in all treatments. No unusual behaviours were noted. No statistically significant difference in length or weight between control and treatment were found. Based on the findings, the 93-day NOEC for hatching success, survival and larval growth is determined to be 0.305 mg a.i./L.

Description of key information

All available data was assessed and the study representing the worst-case effect is included here as key. The result can be considered worst-case and is selected for the CSA.

93-d NOEC = 0.305 mg/L, flow-through, Oncorhynchus mykiss, hatching success, survival or larval growth, OECD TG 210 and EPA OPTS 850.1400, Wheeler 2006

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.305 mg/L

Additional information

There are four standard guideline followed or equivalent to standard guideline and GLP compliant studies available for freshwater fish species. The 93-day early life-stage toxicity flow-through test on embryos and larvae of rainbow trout (Oncorhynchus mykiss) is selected to be the key study, because it represents the worst-case effects (i.e. showed the lowest effect value) for freshwater conditions. The mean measured concentration of the test substance were 5.59,18.8, 39.1, 72.2, 160 and 305 µg/L (measured by a LC-MS/MS; nominal concentrations were 9, 19, 38, 75, 150 and 300 µg/L, respectively). In addition, a dilution water control was included in the test. Observations were made on survival of organisms at hatch and survival and growth (wet weight and total length) of larvae.

The results showed that the egg viability of the fertilisation control was 88% (determined after 14 days). This demonstrated the good quality of eggs used. Egg mortality was low across all treatments. Overall hatching success was good ranging between 80 - 93% and 70 - 93%, for the control and the test substance treatments, respectively. There was no statistically significant difference between control and any test substance treatment for survival at hatching or for survival between hatching and test termination. Swim-up began on day 47 and was complete by day 56 in all treatments. No unusual behaviour was noted. No statistically significant difference in length or weight between control and treatment was found. Based on mean measured concentrations , the 93-day NOEC for hatching success, survival and larval growth is determined to be 0.305 mg/L (Wheeler 2006, OECD TG 210, Reliability 1).

 

The other three freshwater studies are supporting studies. The first supporting study was a 21-d juvenile growth test performed with rainbow trout (Oncorhynchus mykiss). The fish were exposed to nominal test substance concentrations of 0 (blank control), 0.75, 1.5, 3, 6, 12 and 24 mg/L (mean measured: ND, 0.65, 1.25, 2.50, 5.04, 10.10 and 21.03 mg/L). The 21-day NOEC and the 21-day LC50 were determined to be 0.65 and 3.2 mg/L, respectively, based on mean measured concentrations (Jenkins 1989, OECD TG 204, Reliability 1). The second supporting study was a 263-d life cycle toxicity study performed with fathead minnow (Pimepales promelas). The fish were exposed to nominal test substance concentrations of 0 (blank control), 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L (mean measured: < LOQ, 0.060, 0.14, 0.25, 0.51 and 0.95 mg/L). Based on mean measured concentrations, the NOEC was 0.51 mg/L. However, since the  effect on the number of spawns/female was marginal and not consistent with  other population-relevant reproductive endpoints, the overall NOEAC is 0.95  mg/L (Cafarella 2009, OPPTS 850.1500, Reliability 1). The third supporting study was a 21-d short term reproduction assay performed with fathead minnow (Pimephales promelas). The fish were exposed to nominal test substance concentrations of 0 (blank control), 0.18, 0.60 and 2.0 mg/L (mean measured: < LOD, 0.16, 0.54 and 2.0 mg/L). Based on the results of this study, there were no significant effects indicative of endocrine activity up to and including the highest concentration tested (2.0 mg/L) (Cafarella 2009, no guideline but similar to the 2007 OECD draft guideline, Reliability 1).