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Sediment toxicity

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Reference
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-09-14 to 2009-10-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (October 2007)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to German Chemikaliengesetz and Directive 88/320/EEC
Analytical monitoring:
no
Vehicle:
yes
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Details of spiking: The respective test item concentration was weighed out and dissolved in 2.5 mL acetone per replicate. Subsequently, the solution was thoroughly mixed with 10 g quartz sand per replicate in a glass vessel. After complete evaporation of the solvent, the dry quartz sand was given to the sediment. Subsequently, the sediment was mixed thoroughly and filled into the test vessels.
- Equilibration time: 2 days prior to insertion of test organisms
- Equilibration conditions: Same as test
- Controls: Controls with test sediment and quartz sand. Vehicle controls with test sediment and quartz sand treated with acetone.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): organic solvent, acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL per replicate
- Evaporation of vehicle before use: Yes, complete evaporation


Test organisms (species):
Lumbriculus variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Lumbriculus variegatus (Müller), Lumbriculidae, Oligochaeta
- Source: Bundesanstalt für Gewässerkunde (BfG), Am Mainzer Tor 1, 56068 Koblenz, Germany
- Age of test organisms (mean and range, SD): Synchronized from cultured organisms 12 days before test insertion
- Breeding conditions: 20 +/- 2 °C, diffuse light (100 - 1000 lx, 16 h photoperiod) dissolved oxygen concentration > 60 % of the air saturation value
- Handling of worms: 12 days before insertion into the test, the worms were synchronized. Therefore, a sufficient amount of worms was taken from the breeding and cutted into half. After regeneration of heads, the worms were taken from the breeding and cutted into half. After regeneration of heads, the worms were inserted into the test.
- Age of animals at beginning of exposure: 12 days after synchronisation, completely regenerated head region. Synchronization was done to minimize uncontrolled regeneration and reproduction during the test.
- Feeding during test
- Food type: Powder of Urtica dioica (Heinrich Klein GmbH & Co KG, 97525 Schwebheim, Germany)
- Amount: 0.5 % Urtica powder
- Frequency: Urtica powder was mixed into the sediment once at test start.


ACCLIMATION
- Acclimation period: 12 days (after synchronization)
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: Same as test
- Feeding frequency: Same as test (Once)
- Health during acclimation (any mortality observed): No mortality observed
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
artificial sediment
Limit test:
yes
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
Day Parameter Nominal test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 1000
0


Total Hardness [mmol/L CaCO3] 2.87 2.92 3.25
28


Total Hardness [mmol/L CaCO3] 2.57 3.08 3.01
Test temperature:
Day Parameter Nominal test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 1000
0Temperature [°C] 19.3 19.1 19.8



7Temperature [°C] 19.8 19.7 19.7


14Temperature [°C] 21.0 21.1 21.2


21Temperature [°C] 19.3 19.5 19.5


28Temperature [°C] 19.8 19.8 20.0


pH:
Day Parameter Nominal test item concentration
[mg/kg sediment dry weight]
Control Solvent Control 1000
0
pH 7.73 7.86 7.88


7
pH 8.31 8.31 8.23

14
pH 8.48 8.46 8.48

21
pH 8.39 8.41 8.38

28
pH 8.07 8.11 8.16

Dissolved oxygen:


7

O2 [mg/L] 8.52 8.59 8.45
14

O2 [mg/L] 8.42 8.38 8.33
21

O2 [mg/L] 8.65 8.61 8.49
28

O2 [mg/L] 8.77 8.69 8.43
Salinity:
Not measured, freshwater conditions
Ammonia:
Water Parameters (Ammonium-N)
Day Nominal test item concentration [mg/kg sediment dry weight]
Control Solvent Control 1000
Ammonium (NH4-N) in mg/L
0 0.292 0.227 0.269
2 1.66 1.55 1.57
5 1.72 1.54 1.55
7 1.54 0.854 1.18
9 0.768 0.552 0.952
12 1.52 0.904 1.23
14 1.77 1.33 1.54
16 2.52 1.83 2.30
19 3.30 2.44 2.84
21 6.55 6.41 6.15
23 3.82 3.49 3.91
26 3.71 3.98 3.84
28 3.60 3.63 4.36
Nominal and measured concentrations:
1000 mg test substance/kg sediment DW
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 250 mL glass beakers, 6 cm diameter
- Sediment volume: 45 g DW per vessel, ca. 2 cm from the ground
- Overlying water volume: 150 mL
- Depth of sediment and overlying water: Sediment depth ca. 2 cm, water depth ca. 10 cm
- Aeration: yes
- Aeration frequency and intensity: Gentle aeration (2 bubbles per second)
- Replacement of evaporated test water, if any: Yes, once weekly


EXPOSURE REGIME
- No. of organisms per container (treatment): 10 per container (60 per treatment)
- No. of replicates per treatment group: 6 replicates per treatment group
- No. of replicates per control / vehicle control: 6 replicates per control/vehicle control
- Feeding regime: Once at test start
- Type and preparation of food: Powder of Urtica dioica mixed into the sediment before insertion of test organisms
- Amount of food: 0.5 %



OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Reconstituted water acc. to OECD 203
Parameters of the Test Medium at Test Start
Oxygen content [mg/L] pH-value Hardness [mmol/L]
Test Medium at test start 9.09 7.49 2.66


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate): 5 % peat air dried and finely ground, 20 % kaolin, 75 % quartz sand, 0.33 % calcium carbonate

Sediment Properties at Test Start
Total Organic Carbon [%] pH-value Moisture [%]
Sediment at test start 1.5 6.94 0.81


OTHER TEST CONDITIONS

Water temperature [°C] Photoperiod [h] Light intensity [lx]
Nominal 20 +/- 2 16 100 - 500
Actual 17.6 - 21.6* 333 ± 60.8



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality and behaviour changes (Day 0-28), reproduction and biomass at test end (28 d)

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
1000 mg test substance/kg sediment DW

- Range finding study
- Not carried out

Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg sediment dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Mortality, behaviour, biomass, reproduction
Details on results:
- Mortality of test animals at end of exposure period: At test end, the total worm number, including adult and descendant worms, did not statistically significantly differ compared to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight. Also, the number of descendant worms and thus the reproduction did not statistically significantly differ to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight after 28 days of exposure
- Total mass of test animals at beginning of test: 10 worms were equal to ca. 9.6 mg dry mass
- Changes in body weigth of live adults: The dry biomass of worms per replicate was not statistically significantly reduced at the limit test item concentration of 1000 mg/kg sediment dry weight compared to the pooled control
For details please refer to the respective table
- No. of offspring produced:
please refer to the respective table
- Morphological abnormalities: None
- Behavioural abnormalities: None
Reported statistics and error estimates:
t-tests were performed for NOEC calculations. Prior to running t-tests a Normality Test and an Equal Variance Test were done. P- values for both Normality and Equal Variance Test are 0.05. The a-value (acceptable probability of incorrectly concluding that is a difference) is a= 0.05.

No mortality of complete adult worms was observed in the controls as well as at the limit test item concentration of 1000 mg/kg sediment dry weight after an exposure for 28 days.

At test end, the total worm number, including adult and descendant worms, did not statistically significantly differ compared to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight. Also, the number of descendant worms and thus the reproduction did not statistically significantly differ to the pooled control at the limit test item concentration of 1000 mg/kg sediment dry weight after 28 days of exposure

Mortality after 28 days of Exposure

Nominal test item concentration

[mg/kg sediment dry weight]

Total Number of Complete Worms

1

2

3

4

5

6

MV ± SD

% Mortality

Control

10

10

10

10

10

10

10 ± 0

0

Solvent Control

10

10

10

10

10

10

1000

10

10

10

10

10

10

10 ± 0

0

  Total Worm Number after 28 days of Exposure

Nominal test item concentration

Total Number of

[mg/kg sediment dry weight]

1

2

3

4

5

6

MV ± SD

% Inhibition

Significance*

Control

42

41

32

35

36

35

38.0 ± 3.19

--

Solvent Control

42

38

40

40

36

39

1000

37

31

39

38

40

41

37.7 ± 3.56

0.88

No

*) Statistical significance compared to pooled control (P< 0.05)

Reproduction after 28 days of Exposure

Nominal test item concentration

Number of descendant worms

[mg/kg sediment dry weight]

1

2

3

4

5

6

MV ± SD

% Inhibition

Significance*

Control

32

31

22

25

26

25

28.0 ± 3.19

--

Solvent Control

32

28

30

30

26

29

1000

27

21

29

28

30

31

27.7 ± 3.56

1.19

No

*) Statistical significance compared to pooled control (P< 0.05)

No difference in the worm behaviour was observed at the limit test item concentration of 1000 mg/kg sediment dry weight compared to the pooled control during the entire study

Worm Behaviour during 28 Days of Exposure

Nominal test item concentration             [mg/kg sediment dry weight]

Effect

Days after insertion (pooled replicates)

0

2

5

7

9

12

14

16

19

21

23

26

28

Control

s)

60

60

60

60

60

60

60

60

60

60

60

60

60

f)

-

+

+

+

+

+

+

+

+

+

+

+

+

Solvent Control

s)

60

60

60

60

60

60

60

60

60

60

60

60

60

f)

-

+

+

+

+

+

+

+

+

+

+

+

+

1000

s)

60

60

60

60

60

60

60

60

60

60

60

60

60

f)

-

+

+

+

+

+

+

+

+

+

+

+

+

S)= worm burrowing in sediment; f)= faeces on sediment surface; -) negative observation +) positive observation:

The dry biomass of worms per replicate was not statistically significantly reduced at the limit test item concentration of 1000 mg/kg sediment dry weight compared to the pooled control .

Worm Dry Biomass after 28 Days of Exposure per Replicate

Nominal test item concentration             [mg/kg sediment dry weight]

Worm dry weight [mg]

Replicate

Significance*

1

2

3

4

5

6

MV ± SD

Control

24.6

28.9

24.1

35.9

36.4

36.8

34.8 ± 9.11

--

Solvent Control

27.7

31.5

29.5

46.8

54.4

40.6

1000

26.1

31.9

22.8

57.3

20.0

48.0

34.4 ± 15.0

No

*) Statistical significance compared to pooled control (P< 0.05)

Validity criteria fulfilled:
yes
Conclusions:
In this study the test item caused no significant effects on mortality, behaviour and reproduction of L. variegatus at the limit test item concentration of 1000 mg/kg sediment dry weight. Also, the worm biomass was not significantly reduced at the limit test item concentration compared to the control.
Thus, the NOEC was determined to be at 1000 mg/kg sediment dry weight.

All validity criteria recommended by the test guidelines were fulfilled.
Executive summary:

The effects of the test item on the oligochaete Lumbriculus variegatus in a water-sediment system were determined at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany , from September 14thto October 15th, 2009. The study was carried out according to OECD Guideline 225 (2007). Test duration was 28 days after insertion of the test organisms. A limit test with the test item concentration of 1000 mg/kg sediment dry weight was performed by spiking the test item into the sediment. Six replicates per control, solvent control and limit test item concentration with ten worms per replicate were set up. Water quality parameters as temperature, pH-value, O2-content, ammonium and total hardness were determined.

After 28 days no mortality or behavioural changes of L. variegatus were observed at the limit test item concentration of 1000 mg/kg sediment dry weight and the controls (Table 1).

Also, L. variegatus reproduced without any significant differences between the pooled control and the limit test item concentration (Table 1).

Accordingly, the worm biomass per replicate was not statistically significantly reduced at the limit test item concentration compared to the pooled control (Table 1).

  

Overall, the NOEC of the test item concerning mortality, reproduction and biomass is determined to be at 1000 mg/kg sediment dry weight (Table 1).

All validity criteria recommended by the test guidelines were fulfilled.

Summary of all Significant Effects

Effects

Test item

Significance of effects

[mg/kg sediment dry weight]

Worm Mortality

> 1000

Worm Behaviour

> 1000

Worm Biomass

> 1000

Worm Reproduction

> 1000

NOEC

(Mortality, Behaviour, Biomass, Reproduction)

1000

Description of key information

In this study the test item caused no significant effects on mortality, behaviour and reproduction of L. variegatus at the limit test item concentration of 1000 mg/kg sediment dry weight. Also, the worm biomass was not significantly reduced at the limit test item concentration compared to the control.

Thus, the NOEC was determined to be at 1000 mg/kg sediment dry weight.

All validity criteria recommended by the test guidelines were fulfilled.

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:
1 000 mg/kg sediment dw

Additional information

For Pigments Yellow 12 and 83 of the Diarylide Yellow group long-term toxicity tests on sediment organisms were performed. Coherently with the grouping approach, no effects were observed at the limit concentration of 1000 mg/kg sediment dry weight (neither mortality, nor reproduction or biomass and behaviour) for both group members.

Therefore, the NOEC equals the applied nominal concentration of 1000 mg/kg sediment dry weight and the Diarylide Yellow Pigments are judged as chronically nontoxic to sediment organisms.