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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-06-23 - 1999-07-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
OECD Principles on Good Laboratory Practice, (as revised in 1997)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
A stock dispersion was prepared for each test interval by adding 5 mg of the test substance into a 250 mL beaker. Approximately 150 mL water was added and the mixture was homogenized with an ultra-turrax and by ultrasonification (30 minutes). The mixture was than transferred into a graduated flask, filled up to a volume of 5 L with water for dilution (= 1 mg test substance / L) and stirred for 48 hours with a magnetic stirrer. After a sedimentation phase of 1 hour 100 mL portions of the test dispersion were collected from the middle of the stock dispersion vessel using a glass pipette and subsequently transferred to the test vessels.
The test vessels were preconditioned with test solution to prevent adsorption of the test substance which perhaps would reduce the test concentration below the solubility limit. The test vessels were filled with test solution (nominal concentration 1 mg/L), gently swirled and then the test solution was discarded.
The aqueous phase of the concentration group was present as slight yellowish clear solution during the entire exposure period. There were no visible particulate matters.
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance was chemically stable in water over the test intervals of 48 and 72 hours, respectively. The concentration values given in this report are based an the nominal concentration. Due to the very low solubility of the substance it can be estimated, that the actual concentration is smaller or equal to 20 µg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna - Clone 5
Origin: Stock culture of Hoechst Marion Roussel Deutschland GmbH, ProTox
Breeding conditions: 100 mL beakers (height 90 mm, diameter 50 mm), filled with 70 mL test water (according to Elendt, M4) served as culture vessels. One daphnid was used per culture vessel. The animals were used for breeding up to an age of 42 days. The reproduction rate and the state of health of the animals were monitored daily apart from weekends. The animals were fed three times a week with monocellular green algae (Scenedesmus subspicatus).
The supplied diet was based an the amount of organic carbon (C). The ration level is 0.2 mg carbon / day / animal.
During the study the daphnids were fed daily including weekends.
The room was illuminated by fluorescent tubes for 16 hours per day. Daphnids with an age < 24 hours were used for the studies.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
not applicable
Hardness:
Concentration group / Control [mmol Ca(2+) + Mg(2+)]
2.0 - 2.5 / 2.1 - 2.5
Test temperature:
Concentration group / Control [°C]
20.5 to 21.5 / 20.7 to 21.4
pH:
Concentration group / Control
7.8 - 8.3 / 7.8 - 8.1
Dissolved oxygen:
Concentration group / Control [mg/L]
8.6 to 9.4 / 8.6 to 9.3
Salinity:
medium according to Elendt M4
Nominal and measured concentrations:
1 mg/L nominal:
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance was chemically stable in water over the test intervals of 48 and 72 hours, respectively. Due to the very low solubility of the substance it can be estimated, that the actual concentration is smaller or equal to 20 µg/L.
Details on test conditions:
The test chambers were 150 mL beakers, each filled with 100 mL test water.

The room temperature was about 21 °C. The room was illuminated by fluorescent tubes in a 16:8 hours day/night cycle.

A measurement of the light intensity at the position of the test vessels in height of the water surface was carried out during the test. The light intensity was 300 lx.

Study groups:
After the test concentrations had been prepared and water parameters were recorded, 1 daphnid was used in each test chamber following randomization plan No. 1999.0207.

The test concentrations were as follows:
Study group 1: Start Jun-23-99, Test chamber No. 1 - 10 Concentration (mg/L): 0 (control)
Study group 2: Start Jun-23-99, Test chamber No. 11 - 20 Concentration (mg/L): 1

Test procedure
The test was conducted in a semistatic system. The daphnids were transferred into fresh prepared test solutions on Monday, Wednesday and Friday. Inspection and feeding of the daphnids took place every 24 hours and involved recording the immobility, the reproduction rate (number of living and dead offspring) and the development of embryos in the brood pouch. Immobile daphnids were removed from the chambers. Those animals not able to swim within 15 seconds after gentle agitation of the test container were considered to be immobile. The pH-value, oxygen content, conductivity and total hardness were measured and recorded once a week at the beginning and at the end of a test interval in one representative test vessel of each concentration group.

Statistics
To find out whether the mean number of living offspring is significantly reduced by substance effects statistical evaluation was carried out. On the premises of a monotonous concentration-effect-relationship the Wilcoxon-Test was applied. The toxic effect is supposed to be a reduction in the mean number of living offspring per parent animal. The concentration group was compared to the control group.




Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
lmmobility:
No immobility occurred in the 1 mg/L (nominal concentration) and in the control group.

Reproduction rate:
No differences in the onset of brood production were observed in the concentration group in comparison to the control. The reproduction rate in the concentration group showed no statistically significant changes in comparison to the control.

Results with reference substance (positive control):
not applicable

Reproduction rate:

 

control

1 mg/L

n

10

10

mean of living offspring

126.0

115.4

standard deviation

19.26

17.98

coefficient of variation

15.3

15.6

median

124.0

117.5

n = number of daphnids exposed per concentration

Validity criteria fulfilled:
yes
Remarks:
- The mortality of the parent animals did not exceed 20 % at the end of the test in the controls. - The mean number of live offspring produced per parent animal surviving at the end of the test was >= 60.
Conclusions:
In this prolonged toxicity study with the test item in Daphnia magna over 21 days the following effect concentrations were obtained:
NOEC (immobility) = 1 mg/L (nominal concentration, solubility limit <= 20 µg/L)
NOEC (reproduction rate) = 1 mg/L (nominal concentration, solubility limit <= 20 µg/L)
No adverse effects of C.I. Pigment Yellow 13 on Daphnia magna were observed at the maximum tested concentration of 1 mg/L (nominal concentration) which was above the solubility limit (<= 20 µg/L) of the test substance in the test water.
Executive summary:

The test item was tested in Daphnia magna (water flea) over 21 days in a semistatic system (GLP, key). Since no effects were expected up to the limit of solubility a limit test with a nominal concentration of 1 mg/L was conducted.

A negative control (0 mg/L) and a nominal concentration of 1 mg/L were tested. The tested nominal concentration is already above the limit of solubility of the test substance in water ( <= 20 µg/L).

The test was conducted under semistatic conditions. The media and the test solutions were renewed every Monday, Wednesday and Friday. For every renewal new test solutions were prepared. This was done by stirring the test substance for 48 hours in the test medium. The aqueous phase of the concentration group was present as a slightly yellowish clear solution during the entire exposure period. There were no visible particulate matters. Water parameters were within the normal range.

Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test intervals of 48 and 72 hours, respectively.

The concentration values given in this report are based on the nominal concentration.

In the control group no mortality occurred and the mean number of living offspring produced per parent animal was 126.0, thus fulfilling the validity criteria for the test.

No immobility occurred in the 1 mg/L (nominal concentration) and in the control group. No difference in the onset of brood production was observed in the concentration group in comparison to the control. The reproduction rate in the concentration group showed no statistically significant changes in comparison to the control.

In this prolonged toxicity study with C.I. Pigment Yellow 13 in Daphnia magna over 21 days the following effect concentrations were obtained:

NOEC (immobility) = 1 mg/L (nominal concentration, solubility limit <= 20 µg/L)

NOEC (reproduction rate) = 1 mg/L (nominal concentration, solubility limit <= 20 pg/L)

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-09 to 2009-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to German Chemikaliengesetz and Directive 88/320/EEC
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (10 mg/L test item) was prepared with dilution water two days prior to application. The stock solution was shaken with 20 rpm for 48 h. Undissolved particles were removed by centrifugation with 10000 rpm for 10 min.
- Eluate: Dilution water
- Differential loading: Saturated solution as limit concentration
- Controls: 10 replicates of dilution water without test item.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
Not observed
Hardness:
Total Hardness as CaCO3 [mg/L]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 170 182 177 178 175 174
Control 175 185 177 198 180 173
Test temperature:
Temperature [°C]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 22.0 20.8 21.9 20.7 21.9 20.6
Control 21.2 21.1 21.8 21.0 21.8 20.6
pH:
pH-Values
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 7.54 7.73 7.70 7.54 7.74 7.64
Control 7.87 7.77 7.97 7.85 7.94 7.66
Dissolved oxygen:
Dissolved Oxygen Concentration [mg O2/L]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 8.05 8.78 8.62 8.56 7.62 6.90
Control 8.10 9.24 8.04 8.11 8.10 6.48
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to information in materials and methods incl. tables
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): slightly closed with small glass plates
- Material, size, headspace, fill volume: Glass beakers (5 x 8 cm), 100 mL capacity, 50 mL fill volume
- Aeration: No additional aeration
- Renewal rate of test solution (frequency/flow rate): semi-static, medium renewal 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Total organic carbon: not determined
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: Not necessary
- Photoperiod: 16:8 h light:dark cycle
- Light intensity: max. 20 µExm-2xs-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Dissolved oxygen concentration, pH-values, total hardness and water temperature were measured once every 7 days, in fresh and old media, in one replicate of the control and the limit concentration.
Mortality of parental daphnids
The number of parental daphnids which were immobilized or dying during the test and day of death was observed and recorded once a day at least at the same time as the offspring is counted. Dead specimens were removed.
Neonates
First appearance of juveniles was checked daily.
The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of production of neonates until the end of the test. The neonates (alive and dead) were removed after counting to prevent from consuming food intended for the adults. The number of aborted eggs and dates of observation was recorded.
Condition of parental daphnids
Abnormalities (e.g. swimming behaviour, number of males and winter eggs) were observed and recorded at each day of observation.
Total length and dry body weight of parental daphnia
At the end of the test total length of each daphnid and the mean dry body weight of the daphnids alive at the saturated solution and control were determined. The mean dry body weight of a surviving daphnid was calculated from the dry mass of all daphnids alive at the saturated solution and control.

VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
- Test concentrations: 1:1, 1:10, 1:100
- Results used to determine the conditions for the definitive study:
Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Dilution level
of the
saturated solution IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
1:1# 0 0 0 0 0 0
1:10 10 0 5 10 0 5
1:100 0 0 0 0 0 0
Control 0 0 0 0 0 0
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 10 other: mg/L (nominal loading) saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: No adult mortality or immobilization of parental daphnids was observed at the control group and at the saturated solution
- No. of offspring produced per day per female: The mean number of offspring alive produced per parent animal surviving at the end of the test was 116 juveniles at the control group and 117 juveniles at the saturated solution after 21 days. The reproductive output at the limit concentration is comparable to the control group
- Body length and weight of parent animals: please refer to the respective table
- Type and number of morphological abnormalities: no morphological abnormalities detected
- Type and number of behavioural abnormalities: no behavioural abnormalities detected
- Number of males and females (parental): No males were observed in either the control or the test group during the test.
- Time to first brood release or time to hatch: The first day of appearance of juveniles at the test and control group was day 8
- Other biological observations: No ephippia were observed in the control or in the test group during the test. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was < 1 %, which is seen to be not biologically significant. At the control no dead juveniles or aborted eggs were observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.67 (CI 1.63 - 1.71)
Reported statistics and error estimates:
NOEC (No observed The highest tested concentration of the test item at which the test
effect concentration) item is observed to have no statistically or biologically significant effect when compared to the control.

LOEC (Lowest observed The lowest tested concentration of the test item at which the test
effect concentration) item is observed to have a statistically or biologically significant effect when compared to the control.

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                        (n = 10)

Nominal Loading
of the
Saturated Solution
[mg/L]

Mortality [%]

7 days

14 days

21 days

10.0

0

0

0

Control

0

0

0

Number of Juveniles in the Control and Test Group after 21 Days

Nominal Loading of the Saturated
Solution

[mg/L]

No.

Mean No. of Juveniles

Compared

Number of Juveniles in Replicate No.

Total

of

CV

versus

No.

Parents

per Parent

Control

prod. Juv.

producing Juveniles

Inc.

Sum

N

MV ± SD

[%]

[%]

10.0

117

107

119

121

128

112

108

114

123

116

1165

10

117 ±

 7

6

1

Control

102

124

116

126

115

108

118

121

118

114

1162

10

116 ±

 7

6

--

N= number of parents producing juveniles alive; Inc.= increase of reproductive output

First Appearance of Living Juveniles in the Individual Groups

Nominal Loading
of the
Saturated Solution
[mg/L]

Day of First Appearance of Living Juveniles

First

in Replicate No.

Appearance

1

2

3

4

5

6

7

8

9

10

Mean Day

10.0

9

8

8

8

9

8

9

9

8

8

8.4

Control

8

8

8

8

8

8

8

8

8

8

8.0

Total Length and Dry Weight of the Parental Daphnids

Nominal Loading
of the
Saturated Solution
[mg/L]

Total Length of the Parental Daphnids [mm]

MV

N

Dry Weight [mg]

Replicate No.

1

2

3

4

5

6

7

8

9

10

[mm]

S

MV

10.0

5.00

5.50

5.25

5.00

5.75

5.50

5.25

5.50

5.50

5.25

5.35

10

8.6

0.86

Control

5.50

5.50

5.00

5.75

5.50

5.00

5.75

5.25

5.00

5.50

5.38

10

8.7

0.87

S= sum; N= number of parental daphnids alive;

Validity criteria fulfilled:
yes
Conclusions:
At the saturated solution of the test item with a nominal loading of 10.0 mg/L no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were assessed at:
NOEC: Saturated Solution (with the nominal loading of 10.0 mg/L)
LOEC: > Saturated Solution (with the nominal loading of 10.0 mg/L)
All effect values given are based on the saturated solution of the test item with the nominal loading of 10 mg/L.
Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) with the test item was conducted as a limit test with the saturated solution* according to OECD 211 (2008) from 2009-11-09 to 2009-12-03 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.
Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held were used for the limit concentration and control. At test start they were 2 to 24 hours old. The test method was semi-static. The test solutions were renewed 3 times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects on parameters such as occurrence of aborted eggs and stillborn juveniles, time of production of first brood, adult mortality, intrinsic rate of natural increase, dry body weight and length of the parent animals.
A specific analytic of the test item
was not carried out, due to the low solubility of the test item in water. All results given are based on the saturated solution* with the nominal loading of 10 mg/L.

·   The average number of juveniles per parental daphnid in the control group was 116 and 117 at the saturated solution* after 21 days. The reproductive output at the limit concentration is comparable to the control group (Unpaired t-test, p = 0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 6 % at the control group and the saturated solution*.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The IR of the saturated solution* was comparable to the control group (Unpaired t-test, p = 0.05).

·   No adult mortality or immobilization of parental daphnids was observed at the control group and at the saturated solution*.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was lower than 1 % at the saturated solution, which is seen to be not biologically significant. At the control no dead juveniles or aborted eggs were observed.

Five broods were observed during the test period at all surviving parental daphnids. The first brood was released till day 9 by all parent animals of the control and the saturated solution*.

·   The mean dry body weight and total body lengths of all parental daphnids of the test and control group determined at the end of the study were comparable.
The mean dry body weight of the parental daphnids was 0.87 mg per daphnid at the control group and 0.86 mg per daphnid at the saturated solution. The mean body length of the parental daphnids at the saturated solution was 5.35 mm per daphnid and 5.38 mm per daphnid at the control group.

·   No males and ephippia (winter eggs) were observed at control or test groups.

A summary of all test item related effects is given in Table 1. There was no biologically or statistically significant effect determined at the saturated solution* of the test item.

Table 1:    Summary of all Test Item Related Effects

                   (Based on the saturated solution* of test item with the nominal loading of 10 mg/L)

Effects

Test item

Significance

Control

Saturated Solution*

(Nominal Loading 10 mg/L)

Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)

116 ± 7

117 ± 7

no

Coefficient of Variation of the Mean Number of Juveniles per Producing Parent

    6

    6

no

Intrinsic Rate of Natural Increase

    0.49

    0.47

no

Appearance of First Brood [Mean Day]

    8.0

    8.4

no

Number of Broods

    5

    5

no

Stillborn Juveniles and Aborted Eggs

    0

  10

no

Adult Mortality after 21 days [%]

    0

    0

no

Parent Animals: Mean Dry Weight [mg]

    0.87

    0.86

no

Parent Animals: Mean Body Length [mm]

    5.38

    5.35

no

NOECReproduction

Saturated Solution*

(Nominal Loading 10 mg/L)

LOECReproduction

> Saturated Solution*

(Nominal Loading 10 mg/L)

The water quality parameters (pH-value, dissolved oxygen, water hardness and temperature) were determined to be within the acceptable limits.

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromateas reference item once per month.
The EC50 of the reference item at 1.67 mg/L after 24 hours was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to
AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202. *) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium , acc. to OECD Series No. 23 2000(6)

Description of key information

No toxicity of Pigments Yellow 13 and 83, the structural analogues of Pigment Yellow 12, was found in reliable (category 1) OECD 211-tests (Daphnia magna Reproduction Test) with nominal concentrations well above the solubility limit.

Therefore, the Diarylide Yellow Pigments are to be regarded as chronically nontoxic to invertebrates.

Key value for chemical safety assessment

Additional information