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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
Dilution water
In the interests of national and international standardiza-
tion, an artificial medium (synthetic fresh water) (DIN--
German Institute of Standardization, 1982a, b) of the
following composition was used in the test and control
preparations:
tl .76 g CaCl 2. 2H20 (A.R.)/I litre deionized water
4.93 g MgSO 4. 7H20 (A.R.)/I litre deionized water
2.59 g NaHCO 3 (A.R.)/1 litre deionized water
0.23 g KC1 (A.R.)/1 litre deionized water.
Twenty-five millilitres of each solution was pipetted into
a graduated flask and completed to I litre with deionized
water. The amount of calcium and magnesium ions in this
solution was 2.5mmoll -t. The molar relationship of
sodium to potassium 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/a S cm-~, the dilution water was diluted
with 10% tap water.

Stock prepared at 4000 mg/L
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 had been filled with at least 1.61. 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 had been
left to stand for 24 h. Before collecting the water, the tap was
turned on fully and left to run for at least I 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. Nine g 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 fluorescent 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 concen-
trated using the 0.315 mm DIN sieve and separated accord-
ing 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 24 h-old animals on the potential
preparation days in a 21 d test series---Wednesdays 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Details on test conditions:
Dilution water
In the interests of national and international standardiza-
tion, an artificial medium (synthetic fresh water) (DIN--
German Institute of Standardization, 1982a, b) of the
following composition was used in the test and control
preparations:
tl .76 g CaCl 2. 2H20 (A.R.)/I litre deionized water
4.93 g MgSO 4. 7H20 (A.R.)/I litre deionized water
2.59 g NaHCO 3 (A.R.)/1 litre deionized water
0.23 g KC1 (A.R.)/1 litre deionized water.
Twenty-five millilitres of each solution was pipetted into
a graduated flask and completed to I litre with deionized
water. The amount of calcium and magnesium ions in this
solution was 2.5mmoll -t. The molar relationship of
sodium to potassium 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/a S cm-~, the dilution water was diluted
with 10% tap water.
Reference substance (positive control):
not specified
Duration:
24 h
Dose descriptor:
other: EC0
Effect conc.:
558 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
983 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOEC
Remarks:
nominal value
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Remarks:
minimum value
Effect conc.:
10.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
The validity criteria were met as follows.
Altogether, 64 control solutions in beakers and 29
control solutions in closed glass bottles with ground-
glass stoppers were prepared. The reproduction rate
per parent animal after 21 days, in the case of the test
preparation in the beakers, was 88.8 offspring
(SD = 13.1; coefficient of variation = 14.8%), in the
bottles it was 68.0 offspring (SD = 10.6%; coefficient
of variation = 15.6%). The "parent animal mortal-
ity" after 21 days was 7.1% in the case of the test
preparation in beakers and 9.1% in bottles. The "first
offspring" appeared in both types of vessel on the 7th,
and only in a few cases, on the 8th test day at the very
latest. Both met the quality criteria.
The oxygen content and pH value of the test and
control media can influence the test organisms and
thus, they were measured on each transfer day.
Evaluation of the measured data revealed the follow-
ing. 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.
Reported statistics and error estimates:
STATISTICAL EVALUATION OF THE RESULTS
The parameters for fixing the NOEC of the sub-
stance were the mortality of the parent animals, the
reproduction rate and the appearance of the first
offspring during the test period. The Student's t-test
and the U-test were the statistical methods used to
calculate the first two parameters (Sachs, 1969), The
U-test did not require the normal distribution of the
test results and was therefore more suitable for
evaluating the 21 d Daphnia reproduction test. The
highest concentration level, which did not differ
(NOEC) from the control, and the lowest, which did
differ from the control in respect of mortality of the
parent animals and of the reproduction rate, could be
ascertained from the calculations. Moreover, in order
to determine the NOEC, observations and evalua-
tions were also made of the appearance of the first
offspring in the test preparations in comparison with
the control preparations. Primarily, the results were
expressed with reference to the nominal concen-
tration. If, however, the chemical analysis showed a
loss of the tested substance of more than 20%, then
the lowest analysed concentration (minimum value)
obtained during the test was also given for the
NOEC. The aim was to counteract the customary
discrepancies between interpretation and the repro-
ducibility of chemical measured data which move
within a range of variance.
Validity criteria fulfilled:
yes
Conclusions:
The 24 hour EC50 was determined to be 983 mg/L and the 21 day NOEC, based on reproduction was determined to be 16 mg/L nominal

Description of key information

The 24 hour EC50 was determined to be 983 mg/L and the 21 day NOEC, based on reproduction was determined to be 16 mg/L nominal

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
16 mg/L

Additional information