[No public or meaningful name is available]

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 21th december 2010 to 6th april 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test solutions when freshly prepared (the fresh test media) and immediately prior to renewal (the aged test media) a the sart of the study (0 d) and weekly thereafter (2, 8, 16, 28 d). However, only the highest and lowest test concentrations were analyzed unless where the concentration of the test substance is demonstrated not to remain within +/- 20% of the initial analyzed concentration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method :
The stock solution I of the test item (1000 mg/l) for the test was prepared by dissolving 0.1002 g of the test item into 100 ml distilled water. Ultrasonification for 30 min was used to facilitate the dispersion of the test item.
The stock solution II of the test item (100 mg/l) for the test was prepared by drawing 10 ml stock solution I and diluting it to 100 ml with distilled water.
The test solutions were gained by diluting the stock solution with test water. Ultrasonification for 30 min was used to facilitate the dispersion of the solutions.

- Control : dilution medium without test item

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name : Brachydanio rerio (Zebrafish)
- Strain : /
- Source : Commercial fish supplier of Guangzhou Hongfa Aquatic Animals Culture Company in China.
- Age at study initiation : Juvenile
- Length at study initiation : no data
- Weight at study initiation : a subsample of 10 fishes before the test were weighed individually as wet weights with the precision of 1 mg. Mean body weight was 0.064 g ranging from 0.052 g to 0.069 g.
- Method of breeding : feeding fry were held for 14 days in holding tanks supplied with a continuous flow of aerated water before usede for testing.
- Feeding : ???
- Food type : the fish was fed daily with live food (Artemia)
- Amount : the quantity of food being kept constant 2% to 4% dry weight related to the initial fish weight. Food was withheld from the stock population for 24h prior to the start of the test.
- Frequency : daily

ACCLIMATATION
- Acclimation period : 14 days
- Acclimatation conditions :
- Light : 16 hrs photoperiod daily (light intensity : 1000 to 1500 lux)
- Temperature : 23°C to 24°C for Zebrafish
- Oxygen concentration : 90% of the air saturation
- Type and amount of food : live food (Artemia). The quantity of food being kept constant 2% to 4% dry weight related to the initial fish weight. Food was withheldfrom the stock population for 24h prior to the start of the test.
- Type and amount of food : live food (Artemia). the quantity of food being kept constant 2% to 4% dry weight related to the initial fish weight. Food was withheld from the stock population for 24h prior to the start of the test.
- Feeding frequency : daily
- Health during acclimatation :During the holding period the tanks was inspected daily and any debris or unhealthy or dead fish were removed. Fish received no treatment for disease in the two weeks preceding the test and during the test.
- Feed frequency : daily
- Health during acclimation : Any debris or unhealthy or dead fish were removed. Fish received no treatment for disease in the two weeks preceding the test and during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

7 days ??

Hardness:

122.2 mg/L as CACO3 (tap water)

Test temperature:

Measurements of the temperture were carried out at the beginning and end of water renewal.
During the whole test period, the test temperature of the test media was maintained in the range of 22°C to 23°C.

pH:

Measurements of the pH were carried out at the beginning and end of water renewal.
During the whole test period, the pH values of the control medium and test media were between 7.4 and 7.5.

Dissolved oxygen:

Measurements of the dissolved oxygen were carried out at the beginning and end of water renewal.
During the whole test period, the Dissolved Oxygen (DO) values of the control mediums and test media varied from 94% and 98% of the air saturation at the test temperature.

Salinity:

no data

Nominal and measured concentrations:

The following nominal concentrations of the test item were tested : 0.10, 0.25, 0.50, 1.00, 2.50 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel : Tanks made of chemically inert material, with a sealable inert lid, and with a capacity of approximately 8 L.
- Fill volume : 10 liters for each treatment and control
- Aeration : no aeration
- Renewal rate of test solution (frequently : flow rate) :24-h renewal condition
- No. of organisms per vessel : 10 fishes
- No. of vessels per concentration (replicates) : 1
- No. of vessels per control (replicates) : 1
- Biomass loading rate : 0.1 to 1.0 g of fish/L

TEST MEDIUM / WATER PARAMETERS
- Source / preparation of dilution water :
Good quality tap water which had been dechlorinated for at least 24h was used.
- Culture medium different from test medium : no
- Intervals of water quality measurement : Characteristics of the dilution water are measured at leact twice a year by Jiangsu Provincial Center for Disease Prevention and Control. According to the latest measuring results :
- total hardness of the test water : 122.2 mg (CACO3)/L
- oxygen concentration : about 98% of the air saturation value
- pH : 7.55 at room temperature

OTHER TEST CONDITIONS :
- Adjustment of pH : no
- Photoperiod : 16 hrours photoperiod
- Light intensity : 1000 to 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The fish was examined daily during the test period and any external abnormalities (such as hemorrhage, discoloration) and abnormal behavior (such as inactivity, strange swimming pattern, other abnormal behaviour,..) were noted.
Fish were considered dead if there was no visible movement and if touching of the caudal peduncle no reaction. The dead fish were removed as soon as possible, if any.
Representative samples of 10 individuals in the tank population (blotted dry) were weighed before the test starts. Meanwhile, the total wet-weights (blotted dry) of fishes in each concentration were also weighted. At the end of the test, all surviving fish were weighted as wet weights (blotted dry) by individual.
A "pseudo" growth rate of each test fish was calculated for the study period of 28 days, using the equation :
r = [(ln w2 - ln mw1) / (t2 - t1)] x 100
where:
r = ‘pseudo’ specific growth rate of the test fish during the test period
ln w2 = natural logarithm of body weight of each individual fish at the end of the test period
ln mw1 = mean value of the natural logarithms of body weight of all fish in the aquarium at the start of the test period
t1, t2 = day at the start and end of the test period, respectively

RANGE- FINDING STUDY
- Range-finding study : no

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 2.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: mortality, observations for visible abnormalities, fish weight, and pseudo specific growth rate for body wet weight

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 2.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: mortality, observations for visible abnormalities, fish wet weight, and pseudo specific growth rate for body wet weight

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 2.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

ANALYTICAL RESULTS :
The results indicate that with 24h-renewal of the test solution, the concentration of the test item is consistent throughout the test (deviation within 20% of the initial analyzed concentration).Thus, the test item was stable during the test medium renewal period of 24 hours.

BIOLOGICAL RESULTS :
All fishes in the control and the treatment were alive and appeared normal. In summary, no mortality or abnormal behavior was observed in zebrafish at the test concentration of 0.1, 0.25, 0.5, 1 and 2.5 mg/L (nominal concentration).

At the start of the test, fishes weighted about 0.064 g (+/- 8.8%)in average were chose and randomly put into different test solutions. A the end of the test, themean weight of fish in the controls increased more than 60%. Based on analysis of variance, the pseudo specific growth rates for each concentration were then compared with the control values using an multiple comparison method of Dunnett's tests.
The results showed that there was no significant difference for pseudo specific growth rates among all test concentrations and the control (p<0.05).

Table 1 - Mortality and visible abnormalities

Nominal concentration (mg/L)	Initial number of fish	Mortality (%) observation
		Day 2	Day 8	Day 16	Day 28
0	10	0/NB	0/NB	0/NB	0/NB
0.10	10	0/NB	0/NB	0/NB	0/NB
0.25	10	0/NB	0/NB	0/NB	0/NB
0.50	10	0/NB	0/NB	0/NB	0/NB
1.00	10	0/NB	0/NB	0/NB	0/NB
2.50	10	0/NB	0/NB	0/NB	0/NB

NB : Normalbehaviours

Table 2 - Individual and Mean Fish weights on day 0

	No. of the subsample fish
	1	2	3	4	5	6	7	8	9	10
Weights (g)	0.066	0.066	0.069	0.052	0.066	0.065	0.069	0.066	0.059	0.057
Min.	0.052 g
Max.	0.069 g
Mean	0.064 g
RSD	8.82%
	Nominal concentration (mg/L)
	0		0.10		0.25		0.50		1.00	2.50
Total weights (g)	0.652		0.653		0.621		0.621		0.640	0.651
Mean weights (g)	0.065		0.065		0.062		0.062		0.064	0.065

Table 3 - Individual and mean fish weights on day 28

No.	Nominal concentration (mg/L)
	0		0.10		0.25		0.50		1.00		2.50
	Weights (g)	Rates^1 (%)	Weights (g)	Rates (%)	Weights (g)	Rates (%)	Weights (g)	Rates (%)	Weights (g)	Rates (%)	Weights (g)	Rates (%)
1	0.125	2.32	0.116	2.05	0.131	2.67	0.121	2.38	0.133	2.61	0.132	2.52
2	0.122	2.24	0.121	2.20	0.122	2.41	0.113	2.14	0.122	2.30	0.112	1.94
3	0.113	1.96	0.126	2.35	0.130	2.64	0.116	2.23	0.132	2.59	0.103	1.64
4	0.112	1.93	0.122	2.23	0.111	2.07	0.121	2.38	0.116	2.12	0.113	1.97
5	0.131	2.49	0.116	2.05	0.102	1.77	0.131	2.67	0.101	1.63	0.112	1.94
6	0.116	2.06	0.113	1.96	0.124	2.47	0.113	2.14	0.121	2.27	0.106	1.74
7	0.102	1.60	0.105	1.70	0.103	1.81	0.106	1.91	0.103	1.70	0.107	1.77
8	0.103	1.63	0.121	2.20	0.124	2.47	0.113	2.14	0.100	1.59	0.121	2.21
9	0.133	2.55	0.111	1.89	0.107	1.94	0.121	2.38	0.121	2.27	0.126	2.36
10	0.121	2.21	0.130	2.46	0.120	2.35	0.116	2.23	0.110	1.93	0.122	2.24
LOEC			>2.50mg/L (analyzed concentration2.52mg/L)
NOEC			>2.50mg/L (analyzed concentration2.52mg/L)

¹Rates : pseudo specific growth rates for the periods from day 0 to day 28

Validity criteria fulfilled:

yes

Remarks:

mortality in control < 10%; increase of weight in control > x 1.5; acceptable pH, temperature and oxygen concentration

Conclusions:

Based on mortality, observations for visible abnormalities, fish wet weight, and pseudo specific growth rate for body weight, the highest concentration tested without toxic effects (NOEC) observed at Brachydanio rerio after an exposure period of 28 days was determined to be greater than 2.50 mg/L (analyzed concentration 2.52 mg/L)
The LOEC (lowest observed effect concentration) was determined to be higher than 2.50 mg/L (analyzed concentration 2.52 mg/L) due to the absence of toxic effects on test fish.
The EC50 (concentration that would cause a 50% variation in growth rate) was determined to be higher than 2.50 mg/L (analyzed concentration 2.52 mg/L) due to the absence of toxic effects on test fish.

Executive summary:

The study met the acceptability criteria prescribed by the Guidelines (Dissolved Oxygen Concentration : no less than 60% of the air saturation value; temperature : 23 +/- 2°C that not differ by more than +/- 1°C between test chambers; the increasing rate of the mean wet-weight of fish : no less than 60% of the initial weight). Therefore, the test was considered valid.

The LOEC (Lowest Observed Effect Concentration) (28 days) was determined to be higher than 2.50 mg/L (analyzed concentration 2.52 mg/L) due to the absence of toxic effects on test fish.

The NOEC (No Observed Effect Concentration) (28 days) was determined to be greater tha 2.50 mg/L (analyzed concentration 2.52 mg/L) based on mortality, observations for visible abnormalities, fish wet weight, and pseudo specific growth rate for body weight.

The EC50 (concentration that would cause a 50% variation in growth rate) (28 days) was determined to be higher than 2.50 mg/L (analyzed concentration 2.52 mg/L) due to the absence of toxic effects on test fish.

In conclusion, the results showed that under valid semi-static conditions (24h-renewal), the LOEC (28 days), the LOEC (28 days), and the EC50 (28 days) are all greater than 2.50 mg/L (analyzed concentration 2.52 mg/L)

Description of key information

The results showed that under valid semi-static test conditions (24h-renewal), the LOEC(28d), NOEC(28d) and the EC50(28d) of RHODIASOLV IRIS to Zebra Fish are greater than 2.50 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.5 mg/L

Additional information

One experimental study, scored as Klimisch 1, is available (Shi L., 2011) and selected as a key study.