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Long-term toxicity to aquatic invertebrates

Administrative data

long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure according to national standards.

Data source

Referenceopen allclose all

Reference Type:
Reference Type:
secondary source

Materials and methods

Test guideline
other: The test was conducted in line with the provisional procedure proposed by the Federal Environmental Agency (Umweltbundesamt) (as of january the 1st 1984).
Principles of method if other than guideline:
Proposed preliminary testing method by the Federal Environmental Agency of Germany (UBA): "Prolonged toxicity test on Daphnia magna (determination of NOEC for reproduction rate, mortality and time of the first appearance of offspring; 21 d)", (1984)
GLP compliance:
not specified

Test material


Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:

In the interests of national and international standardization, an artificial medium (synthetic fresh water) (DINGerman Institute of Standardization, 1982a, b) of the following composition was used in the test and control preparations:
- 11.76g CaCl2*2H2O (A.R.)/ 1 litre deionized water
- 4.93g MgSO4*7H2O (A.R.)/ 1 litre deionized water
- 2.59g NaHCO3 (A.R.)/ 1 litre deionized water
- 0.23g KCl (A.R.)/ 1 litre deionized water

Twenty-five millilitres of each solution was pipetted into a graduated flast and completed to 1 litre with deionized water. The amount of calcium and magnesium ions in this solution was 2.5 mmol L^-1. The molar relationship of sodium to posassium ions was 10:1. This water was aerated up to the water saturation level and the pH value was measured (8.0 +/- 0.2). When using deionized water with a conductivity of < 1 µS cm^-1, the dilution water was diluted with 10% tap water.


Before preparing the dilution series, the substance was fully dissolved (both quantitatively and optically) in dilution water using magnetic stirrers (stock solution).

From the stock solution of the substance to be tested, graduated dilutions with dilution water were produced in the concentration range in which effects were to be expected in accordance with the results from the acute 24 h Daphnia test and a preliminary 3 d Daphnia test (same conditions as in the 21 d reproduction test). The dilution steps corresponded to a ratio of 1 : 2.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia magna strain (IRCHA strain) has been maintained in accordance with the procedure practised since 1978. In each case, 20-30 specimens were placed in forty 2-1. beakers which bad been filled with at least 1.6L Berlin tap water. They provided 24 h-old animals when the offspring were removed daily from the cultures.
For all Daphnia strain cultures, temperature-controlled, dechlorinated and oxygen-saturated tap water (German hardness 16°, pH value 7.6-7.7) was used which bad been left to stand for 24 h. Before collecting the water, the tap was turned on fully and left to run for at least 1 h. All beakers were covered with watch glasses and placed on a white supporting surface. Feeding with dry algae of the Scenedesmus genus took place daily. 9g of feed were suspended in 1000 ml tap water and 2 ml of the suspension were added to each beaker.
The temperature of the culture area was regulated thermostatically at 20°C. Under exclusion of daylight, the area was lit by ftuorescent lamps (Philips TL 65/33W) for 9 h between 7 a.m. and 4 p.m. On Monday and Thursday of each week the tap water in all beakers was renewed as were the beakers themselves on Mondays. On Mondays, the offspring which had appeared between Thursday or Friday and Monday were concentrated using the 0.315 mm DIN sieve and separated according to size using the 0.630 mm DIN sieve. Daphnia in the different size categories were used separately for further cultivation.

In order to obtain 24h-old animals on the potential preparation days in a 21d test series - Wednesday or Fridays - it was necessary to remove the offspring from the cultivation beakers on Tuesday and/or Thursday. The daphnids which were at most 24 h old were removed by pipette and concentrated on a 0.25 mm DIN sieve, placed in as small an amount of dilution water as possible and used as test organisms.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
21 d

Test conditions

Nominal and measured concentrations:
Tested concentration range: 0.125 - 16.0 mg/L
Details on test conditions:
The semi-static procedure adopted meant that the parent animals in the test and control vessels had to be pipetted 3 times a week (Mondays, Wednesdays and Fridays) into freshly prepared test and control media-in each case at the corresponding concentration level. During this process, dead parent animals or those incapable of swimming were removed. The offspring were counted and the total number for each lest vessel was recorded. Then, the pH value and the oxygen concentration were measured in two test vessels per concentration level. The test and control preparations were observed daily in order inter alia, to record the day on which the first offspring appeared, feeding was carried out at the same time. Tetramin-Hauptfutter (fish feed) and activated sludge were used as feeds. This led to an overall COD of 15-20mg*l^-1 as the daily feed amount. The test culture area was protected from daylight and lit from 7 a.m. to 4 p.m. with fluorescent lamps-Philips TL 40/25W.
Unlike the procedure proposal, the temperature of the test area was set thermostatically at 25 ± 1 °C in order to be sure of meeting the stipulated quality criteria. For reasons of practicability, the chemical determination of the substance concentrations laid down in the test guideline was modified as follows. Samples were taken twice from selected concentration levels of the test series during the lest period and analysed chemically: the first sampling took place on one of the transfer days before the 7th day, i.e. in the period during which no offspring appeared; the second sampling took place between the 16th and 21st day.
For the corresponding dilution levels, the following parameters were determined:
The concentrations of the initial preparations in order to check the solution behaviour and the dilution steps; the concentrations in the test and blank preparations (no test organisms or feed) after an interval of 48/72 h in order to determine the fate of the substance.
Reference substance (positive control):

Results and discussion

Effect concentrations
21 d
Dose descriptor:
Effect conc.:
3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
Details on results:

On no occasion was the pH value-based on 8.0 ± 0.2-lower than 7 .0 in any of the control or test preparations either in beakers or in bottles at the end of the test period, i.e. after 48/72 h; it always remained in the neutral to subalkaline range. Based on the oxygen saturation of the test and control media, an average minimum oxygen saturation value of 69% was measured at the end of the test period in the beakers, and a value of 58% in the bottles. A negative influence on the test organisms could be ruled out even at these extreme values.


For 21d chronic toxicity tests with Daphnia magna the NOEC for reproduction was determined at 3 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
In 21 day chronic toxicity tests with Daphnia magna, the test substance 1-chloro-2-nitrobenzene showed a toxic effect on the reproduction of the test organism with a NOEC of 3 mg/L.
Executive summary:

In 21 day chronic toxicity tests with Daphnia magna, the test substance 1-chloro-2-nitrobenzene showed a toxic effect on the reproduction of the test organism with a NOEC of 3 mg/L.