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 aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Oct 2006 to 27 Oct 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

September 21, 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken from the freshly prepared test media of all test concentrations and the control on Days 0, 7 and 16. To determine the maintenance of the concentrations of the test substance during the test medium renewal periods, duplicate samples were taken from all test media and the control at the end of two test medium renewal periods of 48 hours (on Day 2 and Day 9) and of one test medium renewal period of 72 hours (Day 19). The stability samples were taken from the actual test (containing food) by combining the contents of the test beakers of each test concentration after the end of the treatment period.All samples were deep-frozen (at about -20 °C) immediately after sampling. In a pre-experiment for investigation of the storage stability (without GLP), the test item was determined to be stable in the test water under these storage conditions. The concentrations of the test substance were measured in all duplicate test media samples from the lowest and the highest nominal test concentrations of 0.23 and 2.3 mg/L, respectively. From the control samples, one of the duplicate samples was analysed from each sampling date.

Vehicle:

no

Details on test solutions:

Prior to the start of the test and prior to each test medium renewal, a stock solution of the test item with the nominal concentration of 23 mg/L was freshly prepared by dissolving 23.0 to 23.1 mg of the test item completely in 1000 ml of test water using ultrasonification (15 minutes) and intense stirring for about three hours in the dark. This intensively stirred stock solution was diluted with test water to prepare the test media with concentrations of the test item as mentioned above. The test media were freshly prepared just before introduction of the daphnids (i.e., start of the test) and just prior to each test medium renewal.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Maintain condition: In 1 L glass beakers filled with culture medium and were transferred twice a week to fresh medium.
- Feeding frequency before test: Normally three times a week
- Food type: Green algae of the species Scenedesmus subspicatus and with a fish food suspension.
- Health condition:The health of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.
- Age at study initiation: < 24 hours old (The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny.)
- Feeding during test: The test animals were fed on each working day (Monday through Friday) with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown in the laboratories of the test facility) and a fish food suspension. The fish food suspension was fed in addition to the algal food, because a toxic effect of the test item on the algae could not be excluded. The carbon contents of the algal and fish food suspensions were determined using a analyser. The food amounts were based on the measured concentrations of total organic carbon (TOC) in the food suspensions and consisted of 50% algae and 50% fish food (based on TOC).

The following amounts of TOC were fed per adult test animal and day:
- Day 0/1: 0.10 mg TOC / Daphnia
- Day 2/5-8/12-13: 0.15 mg TOC / Daphnia
- Day 14-15/19-20: 0.20 mg TOC / Daphnia
- Day 9/16: 0.25 mg TOC / Daphnia
- On Day 9 and 16 (Fridays), the amount of food was increased to 0.25 mg TOC per Daphnia to provide a sufficient amount of food for the following two days (Saturday and Sunday).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

19 - 20 °C

pH:

7.7 - 8.0

Dissolved oxygen:

≥ 8.2 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0.023, 0.073, 0.23, 0.73 and 2.3 mg/L
- Mean measured lowest test concentration: 0.019 mg/L
- Mean measured highest test concentration: 2.05 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beaker
- Type: Covered with glass plates
- Aeration: No
- Filled volume: 80 mL of test medium
- Renewal rate of test solution: The test media of all test concentrations and of the control were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period (every Monday, Wednesday, and Friday). In the end, a total of 8 test medium renewals were performed.
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10

TEST MEDIUM
- Source/preparation of dilution water: The test was conducted in reconstituted water with an initial pH of 7.9 ± 0.3. Before use, the test water was aerated until oxygen saturation. During the test, the test media were not aerated.

WATER PARAMETERS
At the beginning and end of each test medium renewal period, the pH values and dissolved oxygen concentrations were measured in one of the beakers of each test concentration and of the control. At the same time periods, the water temperature was measured in one of the control beakers. Additionally, the air-temperature in the testing room was continuously recorded by a temperature recorder. The appearance of the test media was visually recorded at each of the above mentioned intervals.

OTHER TEST CONDITIONS
- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness, with a 30 minute transition period
- Light intensity: A range of about 470 and 640 Lux

EFFECT PARAMETERS MEASURED
- Survival/mobility: In renewed test solution days, the surviving test animals were carefully transferred with the aid of glass tubes from the old test vessels into the freshly prepared test media of the corresponding concentrations.
- Mortality and Body Length of Adults and Number of Offspring: The test replicates were observed for mortality of adults at the start of the test, on the first and second day after treatment and thereafter three times per week before renewal of test media. On the same observation dates, starting from Day 7, the number of offspring was recorded. The test beakers were also checked for the presence of aborted eggs or dead offspring.
- Reproductive parameters: The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test. The body length of the adults was measured at the end of the test by measuring the daphnids from the top of the head to the base of the spine using a binocular microscope.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.023 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

An overview of the results is provided in Table 2 - Table 5 in 'Any other information on results incl. tables'.

- Survival: In the control and at all test concentrations, the survival rate of the test animals at the end of the test was 80% or higher. Thus, the survival rate of Daphnia magna after 21 days was not significantly (> 20%) reduced up to and including the highest test concentration of 2.3 mg/L.

- Offspring production: The first young offspring released from their parent animals were recorded in the control and at the test concentration of 0.073 mg/L on Day 7. At all other test concentrations up to 0.73 mg/L the first young offspring were recorded at observation on Day 9. At the highest concentration tested (2.3 mg/L), no offspring were produced. Since no concentration-effect relationship was determined for the appearance of the first offspring at the test concentrations of 0.023 to 0.73 mg/L, the appearance of the first offspring was considered not to be affected by the test item.

- Reproduction rate: The mean reproduction rate of the daphnids in the control was 77.4 ± 13.8 living offspring per surviving adult (mean ± standard deviation). According to the results of a Williams-test (one-sided smaller, a = 0.05), no significant toxic effect of the test item on the mean reproduction rate was determined at the lowest test concentration of 0.023 mg/L. At the test concentrations of 0.073 to 0.73 mg/L, the mean reproduction rate of the surviving daphnids was statistically significantly reduced to 65 - 75% of the control. At the highest test concentration of 2.3 mg/L reproduction was completely inhibited. The 21-day EC50 for the inhibition of the reproduction rate of the daphnids was calculated to be 0.83 mg/L (95% confidence limits could not be calculated). At the test concentration of 0.23 mg/L, some of the first offspring produced by six of the ten daphnids were dead. This effect was determined only in the first brood and only at this test concentration.

- Body length: The mean body length of the daphnids in the control was 3.81 ± 0.16 mm (mean ± standard deviation). Based on a Williams-test (one sided smaller, a = 0.05), the mean body length was not statistically significantly smaller compared to the control at any test concentration up to and including the highest test concentration of 2.3 mg/L.

Reported statistics and error estimates:

The NOEC and the LOEC were statistically evaluated by testing the mean reproduction rate and the mean body length at the test concentrations for statistically significant differences to the control value by the multiple Williams-test after a one-way analysis of variance (ANOVA). Additionally, the EC50 of the reproduction rate after 21 days was calculated by Moving Average Interpolation.

 Table 2. Number of surviving test animals

 

Exposure day	Nominal concentration of the test item (mg/L)
	Control	0.023	0.073	0.23	0.73	2.3
0	10	10	10	10	10	10
1	10	10	10	10	10	10
2	10	10	10	10	10	10
5	10	10	10	10	10	10
7	10	10	10	10	10	10
9	10	10	10	10	10	10
12	9	10	9	9	10	9
14	9	10	9	9	10	9
16	9	10	9	9	10	9
19	9	10	9	9	10	9
21	9	10	9	8	10	9
% surviving on Day 21	90	100	90	80	100	90

Table 3. The total number of living young daphnids produced by all adults (cumulative values)

Exposure day	Nominal concentration of the test item (mg/L)
	Control	0.023	0.073	0.23	0.73	2.3
0	0	0	0	0	0	0
1	0	0	0	0	0	0
2	0	0	0	0	0	0
5	0	0	0	0	0	0
7	32	0	19	0	0	0
9	135	150	95	115	136	0
12	298	336	250	262	282	0
14	418	416	306	262	282	0
16	473	498	364	320	356	0
19	519	584	461	441	482	0
21	697	698	515	441	577	0
% of control (1)	-	100.1	73.9	63.3	82.8	0.0

(1) based on the value of the last exposure day

Table 4. The number of living offspring produced per surviving adult after 21 days of exposure

 

Replicate No.	Nominal concentration of the test item (mg/L)
	Control	0.023	0.073	0.23	0.73	2.3
1	*	63	64	46 (+4)#	90	0
2	67	91	58	51 (+5)#	41	*
3	87	60	48	56 (+5)#	46	0
4	74	65	*	59 (+6)#	59	0
5	52	61	66	29 (+6)#	68	0
6	95	80	68	50	50	0
7	73	81	56	*	63	0
8	95	69	49	62 (+3)#	58	0
9	80	41	52	*	48	0
10	74	87	54	52	54	0
mean	77.4	69.8	57.2	50.6	57.7	0.0
± SD	13.8	15.1	7.3	10.1	14.0	0.0
n	9	10	9	8	10	9
CV%	17.8	21.6	12.8	20.0	24.3	n.a.
% of control	-	90.1	73.9	65.4	74.5	0.0
STAT	-	n.s.	s.	s.	s.	s.

CV%: coefficient of variation in %: (SDx/Meanx ) · 100%

*:test animal died during the test period

#: dead offspring produced (not taken into account for calculation of the reproduction rates)

n.a.: not applicable

STAT: results of a Williams-test with the mean reproduction rates (one-sided smaller, a= 0.05)

n.s.: mean value not significantly lower than in the control

s.: mean value significantly lower than in the control

Table 5. Body length [mm] of the surviving adults after 21 days of exposure

Replicate No.	Nominal concentration of the test item (mg/L)
	Control	0.0 23	0.073	0.23	0.73	2.3
1	*	3.8	3.8	3.9	3.7	3.8
2	4.0	4.0	3.8	3.8	3.7	*
3	4.1	3.7	3.7	3.8	3.9	3.7
4	3.7	3.8	*	3.7	3.8	3.7
5	3.7	3.7	3.9	3.8	3.7	3.7
6	3.9	3.7	3.9	3.7	4.0	4.0
7	3.6	3.5	3.8	*	3.6	3.8
8	3.8	3.8	3.3	4.0	3.8	3.7
9	3.6	3.5	3.7	*	3.8	3.6
10	3.8	3.6	3.8	3.7	3.7	3.9
mean	3.81	3.71	3.74	3.79	3.78	3.77
± SD	0.16	0.15	0.17	0.10	0.10	0.14
n	9	10	9	8	10	9
CV%	4.3	4.1	4.6	2.7	2.8	3.7
% of control	-	97.4	98.2	99.5	99.1	99.0
STAT	-	n.s.	n.s.	n.s.	n.s.	n.s.

CV%: coefficient of variation in %: (SDx/Meanx ) · 100%

*:test animal died during the test period

STAT: results of a Williams-test with the mean reproduction rates (one-sided smaller, a= 0.05)

n.s.: mean value not significantly lower than in the control

s.: mean value significantly lower than in the control

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 21-day NOEC for survival rates, reproduction rates and body length of the test organism was determined to be 0.023 mg/L.

Executive summary:

 The chronic effect of the test substance on freshwater daphnids (Daphnia magna) was evaluated in a 21-day semi-static toxicity test according to OECD TG 211 and in compliance with GLP criteria. The organisms (1 daphnid/vessel, 10 replicates/concentration) exposed nominal concentrations of 0.023, 0.073, 0.23, 0.73 and 2.3 mg/L. In addition, a negative control was included in the test. The test media were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period. The test replicates were observed for mortality of adults at the start of the test, on the first and second day after treatment and thereafter three times per week before renewal of test media. On the same observation dates, starting from Day 7, the number of offspring was recorded. The test beakers were also checked for the presence of aborted eggs or dead offspring. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test. The body length of the adults was measured at the end of the test by measuring the daphnids from the top of the head to the base of the spine using a binocular microscope. The following test conditions were determined: pH 7.7 - 8.0, 19 - 20 °C and ≥ 8.2 mg/L dissolved oxygen concentration.

After 21 days exposure, the survival rate of the test animals in all groups was ≥ 80%. Thus, the survival rate was not significantly (> 20%) reduced up to and including the highest test concentration of 2.3 mg/L. No concentration-effect relationship was determined for the appearance of the first offspring at the test concentrations of 0.023 to 0.73 mg/L. Thus, the appearance of the first offspring was considered not to be affected by the test item. The mean reproduction rate of the daphnids in the control was 77.4 ± 13.8 living offspring per surviving adult (mean ± standard deviation). No significant toxic effect of the test item on the mean reproduction rate was determined at 0.023 mg/L. At the test concentrations of 0.073 to 0.73 mg/L, the mean reproduction rate of the surviving daphnids was statistically significantly reduced to 65 - 75% of the control. At the highest test concentration of 2.3 mg/L reproduction was completely inhibited. The mean body length of the daphnids in the control was 3.81 ± 0.16 mm (mean ± standard deviation).was not statistically significantly smaller compared to the control at any test concentration up to and including the highest test concentration of 2.3 mg/L. Based on the findings, the 21-d NOEC was determined at 0.023 mg/L (based on reproduction).

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.

Freshwater 21-d NOEC = 0.023 mg/L, semi-static, Daphnia magna, immobility, reproduction, OECD TG 211, Bätscher 2006.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.023 mg/L

Additional information

The chronic effect of the test substance on freshwater daphnids (Daphnia magna) was evaluated in a 21-day semi-static toxicity test according to OECD TG 211 and in compliance with GLP criteria. The organisms (1 daphnid/vessel, 10 replicates/concentration) exposed nominal concentrations of 0.023, 0.073, 0.23, 0.73 and 2.3 mg/L. In addition, a negative control was included in the test. The test media were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period. The test replicates were observed for mortality of adults at the start of the test, on the first and second day after treatment and thereafter three times per week before renewal of test media. On the same observation dates, starting from Day 7, the number of offspring was recorded. The test beakers were also checked for the presence of aborted eggs or dead offspring. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test. The body length of the adults was measured at the end of the test by measuring the daphnids from the top of the head to the base of the spine using a binocular microscope. The following test conditions were determined: pH 7.7 - 8.0, 19 - 20 °C and ≥ 8.2 mg/L dissolved oxygen concentration.

 After 21 days exposure, the survival rate of the test animals in all groups was ≥ 80%. Thus, the survival rate was not significantly (> 20%) reduced up to and including the highest test concentration of 2.3 mg/L. No concentration-effect relationship was determined for the appearance of the first offspring at the test concentrations of 0.023 to 0.73 mg/L. Thus, the appearance of the first offspring was considered not to be affected by the test item. The mean reproduction rate of the daphnids in the control was 77.4 ± 13.8 living offspring per surviving adult (mean ± standard deviation). No significant toxic effect of the test item on the mean reproduction rate was determined at 0.023 mg/L. At the test concentrations of 0.073 to 0.73 mg/L, the mean reproduction rate of the surviving daphnids was statistically significantly reduced to 65 - 75% of the control. At the highest test concentration of 2.3 mg/L reproduction was completely inhibited. The mean body length of the daphnids in the control was 3.81 ± 0.16 mm (mean ± standard deviation).was not statistically significantly smaller compared to the control at any test concentration up to and including the highest test concentration of 2.3 mg/L. Based on the findings, the 21-d NOEC was determined at 0.023 mg/L (based on reproduction).